Boronic ester and acid compounds, synthesis and uses

ABSTRACT

Disclosed herein is a method for reducing the rate of degradation of proteins in an animal, comprising contacting cells of the animal with certain boronic ester and acid compounds. Also disclosed herein are novel boronic ester and acid compounds, their synthesis and uses.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 10/392,165,filed on Mar. 19, 2003, now U.S. Pat. No. 6,747,150, which is acontinuation of application Ser. No. 10/125,997, filed on Apr. 19, 2002,now U.S. Pat. No. 6,617,317, which is a continuation of application Ser.No. 10/100,295, filed on Mar. 18, 2002, now U.S. Pat. No. 6,548,668,which is a continuation of application Ser. No. 09/953,540, filed onSep. 14, 2001, now U.S. Pat. No. 6,465,433, which is a continuation ofapplication Ser. No. 09/490,511, filed on Jan. 25, 2000, now U.S. Pat.No. 6,297,217, which is a division of application Ser. No. 09/085,404,filed on May 26, 1998, now U.S. Pat. No. 6,066,730, which is a divisionof application Ser. No. 08/549,318, filed on Oct. 27, 1995, now U.S.Pat. No. 5,780,454, which is a continuation-in-part of application Ser.No. 08/442,581, filed on May 16, 1995, now U.S. Pat. No. 6,083,903,which is a continuation-in-part of application Ser. No. 08/330,525,filed on Oct. 28, 1994, now abandoned, the contents of each of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to boronic ester and acid compounds, theirsynthesis and uses.

2. Description of Related Art

The synthesis of N-terminal peptidyl boronic ester and acid compounds,in general and of specific compounds, has been described previously(Shenvi et al. U.S. Pat. No. 4,499,082 issued Feb. 12, 1985; Shenvi etal. U.S. Pat. No. 4,537,773 issued Aug. 27, 1985; Siman et al. WO91/13904 published Sep. 19, 1991; Kettner et al., J. Biol. Chem.259(24): 15106–15114 (1984). These compounds have been shown to beinhibitors of certain proteolytic enzymes (Shenvi et al. U.S. Pat. No.4,499,082 issued Feb. 12, 1985; Shenvi et al. U.S. Pat. No. 4,537,773;Siman et al. WO 91/13904 published Sep. 19, 1991; Kettner at al., J.Biol. Chem. 259(24):15106–15114 (1984). A class of N-terminaltri-peptide boronic ester and acid compounds has been shown to inhibitthe growth of cancer cells (Kinder et al. U.S. Pat. No. 5,106,948 issuedApr. 21, 1992). A broad class of N-terminal tri-peptide boronic esterand acid compounds and analogs thereof has been shown to inhibit renin(Kleeman et al. U.S. Pat. No. 5,169,841 issued Dec. 8, 1992).

In the cell, there is a soluble proteolytic pathway that requires ATPand involves covalent conjugation of the cellular proteins with thesmall polypeptide ubiquitin (“Ub”) (Hershko et al., A. Rev. Biochem.61:761–807 (1992); Rechsteiner et al., A. Rev. Cell. Biol. 3:1–30(1987)). Thereafter, the conjugated proteins are hydrolyzed by a 26Sproteolytic complex containing a 20S degradative particle called theproteasome (Goldberg, Eur. J. Biochem. 203:9–23 (1992); Goldberg et al.,Nature 357:375–379 (1992)). This multicomponent system is known tocatalyze the selective degradation of highly abnormal proteins andshort-lived regulatory proteins.

The 20S proteasome is composed of about 15 distinct 20–30 kDa subunits.It contains three different peptidase activities that cleavespecifically on the carboxyl side of the hydrophobic, basic, and acidicamino acids (Goldberg et al., Nature 357:375–379 (1992); Goldberg, Eur.J. Biochem. 203:9–23 (1992); Orlowski, Biochemistry 29:10289(1990);Rivett et al., Archs. Biochem. Biophys. 218:1 (1989); Rivett et al., J.Biol. Chem. 264:12,215–12,219 (1989); Tanaka et al., New Biol. 4:1–11(1992)). These peptidase activities are referred to as thechymotrypsin-like activity, the trypsin-like activity, and thepeptidylglutamyl hydrolyzing activity, respectively.

Various inhibitors of the peptidase activities of the proteasome havebeen reported (Dick et al., Biochemistry 30:2725–2734 (1991); Goldberget al., Nature 357:375–379 (1992); Goldberg, Eur. J. Biochem. 203:9–23(1992); Orlowski, Biochemistry 29:10289 (1990); Rivett et al., Archs.Biochem. Biophys. 218:1 (1989); Rivet et al., J. Biol. Chem.264:12,215–12,219(1989); Tanaka et al., New Biol. 4:1–11 (1992);Murakami et al., Proc. Natl. Acad. Sci. U.S.A. 83:7588–7592 (1986); Liet al., Biochemistry 30:9709–9715 (1991); Goldberg, Eur. J. Biochem.203:9–23 (1992); Aoyagi et al., Proteases and Biological Control, ColdSpring Harbor Laboratory Press (1975), pp. 429–454.

Stein et al., U.S. patent application Ser. No. 08/212,909 filed Mar. 15,1994, describe the use of peptide aldehydes to 1) reduce the rate ofloss of muscle mass in an animal by contacting cells of the muscle witha peptide aldehyde proteasome inhibitor, 2) reduce the rate ofintracellular protein breakdown in an animal by contacting cells of theanimal with a peptide aldehyde proteasome inhibitor, and 3) reduce therate of degradation of p53 protein in an animal by administering to theanimal a peptide aldehyde proteasome inhibitor.

Palombella et al., PCT application serial number PCT/US95/03315, filedMar. 17, 1995, describe the use of peptide aldehydes to reduce thecellular content and activity of NF-κB in an animal by contacting cellsof the animal with a peptide aldehyde inhibitor of proteasome functionor ubiquitin conjugation.

The transcription factor NF-κB and other members of the rel family ofprotein complexes play a central role in the regulation of a remarkablydiverse set of genes involved in the immune and inflammatory responses(Grilli et al., International Review of Cytology 143:1–62 (1993)). NF-κBexists in an inactive form in the cytoplasm complexed with an inhibitorprotein, IκB. In order for the NF-κB to become active and perform itsfunction, it must enter the cell nucleus. It cannot do this, however,until the IκB portion of the complex is removed, a process referred toby those skilled in the art as the activation of, or processing of,NF-κB. In some diseases, the normal performance of its function by theNF-κB can be detrimental to the health of the patient. For example,NF-κB is essential for the expression of the human immunodeficiencyvirus (HIV). Accordingly, a process that would prevent the activation ofthe NF-κB in patients suffering from such diseases could betherapeutically beneficial.

Goldberg and Rock, WO 94/17816, filed Jan. 27, 1994, describe the use ofproteasome inhibitors to inhibit MHC-I antigen presentation. Theubiquitination/proteolysis pathway is shown to be involved in theprocessing of internalized cellular or viral antigens into antigenicpeptides that bind to MHC-I molecules on an antigen presenting cell.Accordingly, inhibitors of this pathway would be useful for thetreatment of diseases that result from undesired response to antigenpresentation, including autoimmune diseases and transplant rejection.

Cyclins are proteins that are involved in cell cycle control ineukaryotes. Cyclins presumably act by regulating the activity of proteinkinases, and their programmed degradation at specific stages of the cellcycle is required for the transition from one stage to the next.Experiments utilizing modified ubiquitin (Glotzer et al., Nature349:132–138 (1991); Hershko et al., J. Biol. Chem. 266:376 (1991)) haveestablished that the ubiquitination/proteolysis pathway is involved incyclin degradation. Accordingly, compounds that inhibit this pathwaywould cause cell cycle arrest and would be useful in the treatment ofcancer, psoriasis, restenosis, and other cell proliferative diseases.

SUMMARY OF THE INVENTION

The present invention provides previously unknown peptidyl boronic acidester and acid compounds. The present invention also provides methods ofusing amino acid or peptidyl boronic ester and acid compounds, ingeneral, as inhibitors of proteasome function.

In a first embodiment, the present invention provides novel boronic acidand ester compounds having formula (1a) or (2a), as set forth below.

An additional aspect of the present invention is related to thediscovery that amino acid and peptidyl boronic acids and esters, ingeneral, are potent and highly selective proteasome inhibitors and canbe employed to inhibit proteasome function. Inhibition of proteasomefunction has a number of practical therapeutic and prophylacticapplications.

In a second embodiment, the present invention provides a method forreducing the rate of muscle protein degradation in a cell comprisingcontacting said cell with a proteasome inhibitor having formula (1b) or(2b) as defined below. This aspect of the present invention findspractical utility in inhibiting (reducing or preventing) the acceleratedbreakdown of muscle proteins that accompanies various physiological andpathological states and is responsible to a large extent for the loss ofmuscle mass (atrophy) that follows nerve injury, fasting, fever,acidosis, and certain endocrinopathies.

In a third embodiment, the present invention provides a method forreducing the activity of NF-κB in a cell comprising contacting the cellwith a proteasome inhibitor of the formula (1b) or (2b), as set forthbelow. The inhibitors employed in the practice of the present inventionare capable of preventing this activation. Thus, blocking NF-κB activityis contemplated as possessing important practical application in variousareas of medicine, e.g., inflammation, sepsis, AIDS, and the like.

In a fourth embodiment, the present invention provides a method ofreducing the rate of degradation of p53 protein in a cell comprisingadministering to the cell a proteasome inhibitor of the formula (1b) or(2b), as set forth below.

In a fifth embodiment, the present invention provides a method forinhibiting cyclin degradation in a cell comprising contacting said cellswith a proteasome inhibitor of the formula (1b) or (2b), as set forthbelow. Inhibiting cyclin degradation is contemplated as possessingimportant practical application in treating cell proliferative diseases,such as cancer, restenosis and psoriasis.

In a sixth embodiment, the present invention provides a method forinhibiting the growth of a cancer cell, comprising contacting said cellwith a proteasome inhibitor of the formula (1a) or (2a), as set forthbelow.

In a seventh embodiment, the present invention provides a method forinhibiting antigen presentation in a cell comprising administering tothe cell a proteasome inhibitor of the formula (1b) or (2b), as setforth below.

In an eighth embodiment, the present invention provides a method forinhibiting inducible NF-κB dependent cell adhesion in an animalcomprising administering to said animal a proteasome inhibitor of theformula (1b) or (2b), as set forth below.

In a ninth embodiment, the present invention provides a method forinhibiting HIV replication in an animal comprising administering to saidanimal a proteasome inhibitor of the formula (1b) or (2b), as set forthbelow.

In a tenth embodiment, the present invention provides an approach forinhibiting cytolytic immune responses. The proteasome inhibitors offormula (1b) or (2b) can be used to inhibit the processing ofinternalized cellular or viral antigens into antigenic peptides thatbind to MHC-I molecules in an animal, and are therefore useful fortreating autoimmune diseases and preventing rejection of foreigntissues, such as transplanted organs or grafts.

In an eleventh embodiment, the present invention provides pharmaceuticalcompositions that comprise compounds of formula (1a), (1b), (2a) or (2b)in an amount effective to inhibit proteasome function in a mammal, and apharmaceutically acceptable carrier or diluent.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Three day cumulative urinary 3-methylhistidine.

FIG. 2. NF-κB binding activity.

FIG. 3. Inhibition by MG-273.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

A first aspect of the present invention is directed to novel subsets ofboronic acid and ester compounds having formula (1a) or (2a) below.Novel compounds of formula (1a) include the following:

or a pharmaceutically acceptable salt thereof;wherein

P is hydrogen or an amino-group-protecting moiety as further definedherein;

B¹, at each occurrence, is independently one of N or CH;

X¹, at each occurrence, is independently one of —C(O)—NH—, —CH₂—NH—,—CH(OH)—CH₂—, —CH(OH)—CH(OH)—, —CH(OH)—CH₂—NH—, —CH═CH—, —C(O)—CH₂—,—SO₂—NH—, —SO₂—CH₂— or —CH(OH)—CH₂—C(O)—NH—, provided that when B¹ is N,then the X¹ attached to said B¹ is —C(O)—NH—;

X² is one of —C(O)—NH—, —CH(OH)—CH₂—, —CH(OH)—CH(OH)—, —C(OH)—CH₂—,—SO₂—NH—, —SO₂—CH₂— or —CH(OH)—CH₂—C(O)—NH—;

R is hydrogen or alkyl, or R forms together with the adjacent R¹, orwhen A is zero, forms together with the adjacent R², anitrogen-containing mono-, bi- or tri-cyclic, saturated or partiallysaturated ring system having 4–14 ring members, that can be optionallysubstituted by one or two of keto, hydroxy, alkyl, aryl, aralkyl, alkoxyor aryloxy;

R¹, at each occurrence, is independently one of hydrogen, alkyl,cycloalkyl, aryl, a 5–10 membered saturated, partially unsaturated oraromatic heterocycle or —CH₂—R⁵, where the ring portion of any of saidaryl, aralkyl, alkaryl or heterocycle can be optionally substituted;

R² is one of hydrogen, alkyl, cycloalkyl, aryl, a 5–10 memberedsaturated, partially unsaturated or aromatic heterocycle or —CH₂—R⁵,where the ring portion of any of said aryl, aralkyl, alkaryl orheterocycle can be optionally substituted;

R³ is one of hydrogen, alkyl, cycloalkyl, aryl, a 5–10 memberedsaturated, partially unsaturated or aromatic heterocycle or —CH₂—R⁵,where the ring portion of any of said aryl, aralkyl, alkaryl orheterocycle can be optionally substituted;

R⁵, in each instance, is one of aryl, aralkyl, alkaryl, cycloalkyl, a5–10 membered saturated, partially unsaturated or aromatic heterocycleor —W—R⁶, where W is a chalcogen and R⁶ is alkyl, where the ring portionof any of said aryl, aralkyl, alkaryl or heterocycle can be optionallysubstituted;

Z¹ and Z² are independently one of alkyl, hydroxy, alkoxy, or aryloxy,or together Z¹ and Z² form a moiety derived from a dihydroxy compoundhaving at least two hydroxy groups separated by at least two connectingatoms in a chain or ring, said chain or ring comprising carbon atoms,and optionally, a heteroatom or heteroatoms which can be N, S, or O; and

A is 0, 1, or 2.

Other novel boronic acid and ester derivatives include compounds havinga single amino acid side-chain. These compounds have the followingformula:

and pharmaceutically acceptable salts thereof;wherein

Y is one of R⁸—C(O)—, R⁸—SO₂—, R⁸—NH—C(O)— or R⁸—O—C(O)—, where R⁸ isone of alkyl, aryl, alkaryl, aralkyl, any of which can be optionallysubstituted, or when Y is R⁸—C(O)— or R⁸—SO₂—, then R⁸ can also be anoptionally substituted 5–10 membered, saturated, partially unsaturatedor aromatic heterocycle;

X³ is a covalent bond or —C(O)—CH₂—;

R³ is one of hydrogen, alkyl, cycloalkyl, aryl, a 5–10 memberedsaturated, partially unsaturated or aromatic heterocycle or —CH₂—R⁵,where the ring portion of any of said aryl, aralkyl, alkaryl orheterocycle can be optionally substituted;

R⁵, in each instance, is one of aryl, aralkyl, alkaryl, cycloalkyl, a5–10 membered saturated, partially unsaturated or aromatic heterocycleor —W—R⁶, where W is a chalcogen and R⁶ is alkyl, where the ring portionof any of said aryl, aralkyl, alkaryl or heterocycle can be optionallysubstituted; and

Z¹ and Z² are independently alkyl, hydroxy, alkoxy, aryloxy, or togetherform a moiety derived from dihydroxy compound having at least twohydroxy groups separated by at least two connecting atoms in a chain orring, said chain or ring comprising carbon atoms, and optionally, aheteroatom or heteroatoms which can be N, S, or O;

provided that when Y is R⁸—C(O)—, R⁸ is other than phenyl, benzyl orC₁–C₃ alkyl.

Alternatively, the group Y in formula (2a) above, can be:

P is one of R⁷—C(O)—, R⁷—SO₂—, R⁷—NH—C(O)— or R⁷—O—C(O)—;

R⁷ is one of alkyl, aryl, alkaryl, aralkyl, any of which can beoptionally substituted, or when Y is R⁷—C(O)— or R⁷—SO₂—, R⁷ can also bean optionally substituted 5–10 membered saturated, partially unsaturatedor aromatic heterocycle; and

R¹ is defined above as for formula (1a).

Pharmaceutical compositions that comprise compounds of formula (1a) or(2a) in an amount effective to inhibit proteasome function in a mammal,and a pharmaceutically acceptable carrier or diluent are within thescope of the present invention.

A second aspect of the present invention lies in the discovery thatboronic acid and ester derivatives of amino acids and peptides, ingeneral, as well as isosteric variations thereof, inhibit proteasomefunction. Thus, the present invention also relates to the use ofproteasome inhibitors having formula (1b) or (2b) for reducing the rateof proteasome dependent intracellular protein breakdown, such asreducing the rate of muscle protein degradation, reducing the rate ofdegradation of p53 protein, and inhibiting cyclin degradation, and forinhibiting the activity of NF-κB in a cell.

Finally, the present invention relates to the use of proteasomeinhibitors having formula (1b) or (2b) for treating specific conditionsin animals that are mediated or exacerbated, directly or indirectly, byproteasome functions. These conditions include inflammatory conditions,such as tissue rejection, organ rejection, arthritis, infection,dermatoses, inflammatory bowel disease, asthma, osteoporosis,osteoarthritis and autoimmune disease such as lupus and multiplesclerosis; cell proliferative diseases, such as cancer, psoriasis andrestenosis; and accelerated muscle protein breakdown that accompaniesvarious physiological and pathological states and is responsible to alarge extent for the loss of muscle mass (atrophy) that follows nerveinjury, fasting, fever, acidosis, and certain endocrinopathies.

Proteasome inhibitors of formula (1b) include:

or a pharmaceutically acceptable salt thereof;wherein

P¹⁰ is hydrogen or an amino-group-protecting moiety;

B¹¹ is independently one of N or CH;

X¹¹, at each occurrence, is independently one of —C(O)—NH—, —CH₂—NH—,—CH(OH)—CH₂—, —CH(OH)—CH(OH)—, —CH(OH)—CH₂—NH—, —CH═CH—, —C(O)—CH₂—,—SO₂—NH—, —SO₂—CH₂— or —CH(OH)—CH₂—C(O)—NH—, provided that when B¹¹ isN, then X¹¹ is —C(O)—NH;

X¹² is one of —C(O)—NH—, —CH(OH)—CH₂—, —CH(OH)—CH(OH)—, —C(O)—CH₂—,—SO₂—NH—, —SO₂—CH₂— or —CH(OH)—CH₂—C(O)—NH—;

R¹⁰ is hydrogen or alkyl or R¹⁰ forms together with the adjacent R¹¹, orwhen A¹⁰ is zero, forms together with the adjacent R¹², anitrogen-containing mono-, bi- or tri-cyclic, saturated or partiallysaturated ring system having 4–14 ring members, that can be optionallysubstituted by one or two of keto, hydroxy, alkyl, aryl, aralkyl, alkoxyor aryloxy;

R¹¹, at each occurrence, is independently one of hydrogen, alkyl,cycloalkyl, aryl, a 5–10 membered saturated, partially unsaturated oraromatic heterocycle or —CH₂—R¹⁵, where the ring portion of any of saidaryl, aralkyl, alkaryl or heterocycle can be optionally substituted;

R¹² and R¹³ are each independently one of hydrogen, alkyl, cycloalkyl,aryl, a 5–10 membered saturated, partially unsaturated or aromaticheterocycle or —CH₂—R¹⁵, where the ring portion of any of said aryl,aralkyl, alkaryl or heterocycle can be optionally substituted,

-   -   where R¹⁵ is aryl, aralkyl, alkaryl, cycloalkyl, a 5–10 membered        saturated, partially unsaturated or aromatic heterocycle, or        -chalcogen-alkyl, where the ring portion of any of said aryl,        aralkyl, alkaryl or heterocycle can be optionally substituted;

Z¹¹ and Z¹² are independently alkyl, hydroxy, alkoxy, aryloxy, or Z¹¹and Z¹² together form a moiety derived from a dihydroxy compound havingat least two hydroxy groups separated by at least two connecting atomsin a chain or ring, said chain or ring comprising carbon atoms, andoptionally, a heteroatom or heteroatoms which can be N, S, or O; and

A¹⁰ is 0, 1, or 2

Proteasome inhibitors of formula (2b) include:

or pharmaceutically acceptable salts thereof;wherein

Y¹⁰ is one of R⁸—C(O)—, R⁸—SO₂—, R⁸—NH—C(O)— or R⁸—O—C(O)—, where R⁸ isone of alkyl, aryl, alkaryl, aralkyl, any of which can be optionallysubstituted, or when Y is R⁸—C(O)— or R⁸—SO₂—, then R⁸ can also be anoptionally substituted 5–10 membered, saturated, partially unsaturatedor aromatic heterocycle;

X¹³ is a covalent bond or —C(O)—CH₂—;

R¹³ is one of hydrogen, alkyl, cycloalkyl, aryl, a 5–10 memberedsaturated, partially unsaturated or aromatic heterocycle or —CH₂—R¹⁵,where the ring portion of any of said aryl, aralkyl, alkaryl orheterocycle can be optionally substituted;

R¹⁵, in each instance, is one of aryl, aralkyl, alkaryl, cycloalkyl, a5–10 membered saturated, partially unsaturated or aromatic heterocycleor —W—R¹⁶, where W is a chalcogen and R¹⁶ is alkyl, where the ringportion of any of said aryl, aralkyl, alkaryl or heterocycle can beoptionally substituted; and

Z¹¹ and Z¹² are independently alkyl, hydroxy, alkoxy, aryloxy, ortogether form a moiety derived from a dihydroxy compound having at leasttwo hydroxy groups separated by at least two connecting atoms in a chainor ring, said chain or ring comprising carbon atoms, and optionally, aheteroatom or heteroatoms which can be N, S, or O.

Alternatively, the group Y in formula (2b) can be:

P is one of R⁷—C(O)—, R⁷—SO₂—, R⁷—NH—C(O)— or R⁷—(O)—C(O)—;

R⁷ is one of alkyl, aryl, alkaryl, aralkyl, any of which can beoptionally substituted, or when Y is R⁷—C(O)— or R⁷—SO₂—, R⁷ can also bean optionally substituted 5–10 membered saturated, partially unsaturatedor aromatic heterocycle; and

R¹ is as defined for formula (1a) above.

Preferred embodiments of the aforementioned methods of use employcompounds of formula (1a) and formula (2a) as defined above.

Pharmaceutical compositions comprising an effective amount of theproteasome inhibitors of formula (2a) or (2b), in combination with anyconventional pharmaceutically acceptable carrier or diluent, areincluded in the present invention.

The term “amino-group-protecting moiety,” as used herein, refers toterminal amino protecting groups that are typically employed in organicsynthesis, especially peptide synthesis. Any of the known categories ofprotecting groups can be employed, including acyl protecting groups,such as acetyl, and benzoyl; aromatic urethane protecting groups, suchas benzyloxycarbonyl; and aliphatic urethane protecting groups, such astert-butoxycarbonyl. See, for example, The Peptides, Gross andMienhoffer, eds., Academic Press, New York (1981), Vol. 3, pp. 3–88; andGreen, T. W. & Wuts, P. G. M., Protective Groups in Organic Synthesis,2nd edition, John Wiley and Sons, Inc., New York (1991). Preferredprotecting groups include aryl-, aralkyl-, heteroaryl- andheteroarylalkyl-carbonyl and sulfonyl moieties.

As used herein, the term “heterocycle” is intended to mean a stable 5-to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclicmoieties that are either saturated or unsaturated, and which consist ofcarbon atoms and from 1 to 4 heteroatoms independently selected from thegroup consisting of N, O and S, wherein the nitrogen and sulfurheteroatoms can optionally be oxidized, the nitrogen can optionally bequaternized, and including any bicyclic group in which any of theabove-defined heterocyclic rings is fused to a benzene ring. Theheterocyclic ring can be attached to its pendant group at any heteroatomor carbon atom that results in a stable formula. The heterocyclic ringsdescribed herein can be substituted on carbon or on a nitrogen atom ifthe resulting compound is stable. Examples of such heterocycles include,but are not limited to, pyridyl, pyrimidinyl, furanyl, thienyl,pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl,benzothiophenyl, indolyl, indolenyl, quinolinyl, isoquinolinyl,benzimidazolyl, piperidinyl, pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl,tetrahydrofuranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl or octahydroisoquinolinyl, azocinyl, triazinyl,6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl, thiophene(yl),thianthrenyl, furanyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl,phenoxathiinyl, 2H-pyrrolyl, pyrrole, imidazolyl, pyrazolyl,isothiazolyl, isoxazolyl, pyridinyl, pyrazinyl, pyrimidinyl,pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl, 1H-indazolyl,purinyl, 4H-quinolizinyl, isoquinolinyl, quinolinyl, phthalazinyl,naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl,4aH-carbazolyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl,phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl,isochromanyl, chromanyl, pyrrolidinyl, imidazolidinyl, imidazolinyl,pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl, isoindolinyl,quinuclidinyl, morpholinyl or oxazolidinyl. Also included are fused ringand spiro compounds containing, for example, the above heterocycles.

The term “substituted”, as used herein, means that one or more hydrogensof the designated moiety are replaced with a selection from theindicated group, provided that no atom's normal valency is exceeded, andthat the substitution results in a stable compound. When a substituentis keto (i.e., ═O), then 2 hydrogens attached to an atom of the moietyare replaced.

By “stable compound” or “stable formula” is meant herein a compound thatis sufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture and formulation into an efficacious therapeuticagent.

The term “heteroaryl” as employed herein refers to groups having 5 to 14ring atoms; 6, 10 or 14 π electrons shared in a cyclic array; andcontaining carbon atoms and 1, 2 or 3 oxygen, nitrogen or sulfurheteroatoms (where examples of heteroaryl groups are: thienyl,benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl, pyranyl,isobenzofuranyl, benzoxazolyl, chromenyl, xanthenyl, phenoxathiinyl,2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl,pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl,indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl,phthalazinyl, naphthyridinyl, quinazolinyl, cinnolinyl, pteridinyl,4aH-carbazolyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl,perimidinyl, phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl,isoxazolyl, furazanyl and phenoxazinyl groups).

The terms “substituted heteroaryl” or “optionally substitutedheteroaryl,” used in reference to R¹, refer to heteroaryl groups, asdefined above, having one or more substituents selected from halogen,C₁₋₆ alkyl, C₁₋₆ alkoxy, carboxy, amino, C₁₋₆ alkylamino and/ordi(C₁₋₆)alkylamino.

The term “aryl” as employed herein by itself or as part of another grouprefers to monocyclic or bicyclic aromatic groups containing from 6 to 12carbons in the ring portion, preferably 6–10 carbons in the ringportion, such as phenyl, naphthyl or tetrahydronaphthyl.

The term “substituted aryl” as employed herein includes aryl groups, asdefined above, that include one or two substituents on either the phenylor naphthyl group selected from C₁₋₆ alkyl, C₃₋₈ cycloalkyl, C₁₋₆alkyl(C₃₋₈)cycloalkyl, C₂₋₈ alkenyl, C₂₋₈ alkynyl, cyano, amino, C₁₋₆alkylamino, di(C₁₋₆)alkylamino, benzylamnino, dibenzylamino, nitro,carboxy, carbo(C₁₋₆)alkoxy, trifluoromethyl, halogen, C₁₋₆ alkoxy, C₆₋₁₀aryl(C₁₋₆)alkoxy, hydroxy, C₁₋₆ alkylthio, C₁₋₆ alkylsulfinyl, C₁₋₆alkylsulfonyl, C₆₋₁₀ aryl, C₆₋₁₀ arylthio, C₆₋₁₀ arylsulfinyl and/orC₆₋₁₀ arylsulfonyl.

The term “alkyl” as employed herein includes both straight and branchedchain radicals of up to 12 carbons, preferably 1–8 carbons, such asmethyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, pentyl,hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl,2,2,4-trimethylpentyl, nonyl, decyl, undecyl and dodecyl.

The term “substituted alkyl” as employed herein includes alkyl groups asdefined above that have one, two or three halo substituents, or one C₁₋₆alkyl(C₆₋₁₀)aryl, halo(C₆₋₁₀)aryl, C₃₋₈ cycloalkyl, C₁₋₆alkyl(C₃₋₈)cycloalkyl, C₂₋₈ alkenyl, C₂₋₈ alkynyl, hydroxy and/orcarboxy.

The term “cycloalkyl” as employed herein includes saturated cyclichydrocarbon groups containing 3 to 12 carbons, preferably 3 to 8carbons, which include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, cyclodecyl and cyclododecyl, any of whichgroups can be substituted with substituents such as halogen, C₁₋₆ alkyl,alkoxy and/or hydroxy group.

The term “aralkyl” or “arylalkyl” as used herein by itself or as part ofanother group refers to C₁₋₆alkyl groups as discussed above having anaryl substituent, such as benzyl.

The term “halogen” or “halo” as used herein by itself or as part ofanother group refers to chlorine, bromine, fluorine or iodine withchlorine being preferred.

For medicinal use, the pharmaceutically acceptable acid and baseaddition salts, those salts in which the anion does not contributesignificantly to toxicity or pharmacological activity of the organiccation, are preferred. Basic salts are formed by mixing a solution of aboronic acid (Z¹ and Z² are both OH) of the present invention with asolution of a pharmaceutically acceptable non-toxic base, such as,sodium hydroxide, potassium hydroxide, sodium bicarbonate, sodiumcarbonate, or an amino compound, such as choline hydroxide, Tris,bis-Tris, N-methylglucamine or arginine. Water-soluble salts arepreferable. Thus, suitable salts include: alkaline metal salts (sodium,potassium etc.), alkaline earth metal salts (magnesium, calcium etc.),ammonium salts and salts of pharmaceutically acceptable amines(tetramethylammonium, triethylamine, methylamine, dimethylamine,cyclopentylamine, benzylamine, phenethylamine, piperidinemonoethanolamine, diethanolamine, tris(hydroxymethyl)amine, lysine,arginine and N-methyl-D-glucamine).

The acid addition salts are obtained either by reaction of an organicbase of formula (1a) or (2a) with an organic or inorganic acid,preferably by contact in solution, or by any of the standard methodsdetailed in the literature available to any practitioner skilled in theart. Examples of useful organic acids are carboxylic acids such asmaleic acid, acetic acid, tartaric acid, propionic acid, fumaric acid,isethionic acid, succinic acid, cyclamic acid, pivalic acid and thelike; useful inorganic acids are hydrohalide acids such as HCl, HBr, HI;sulfuric acid; phosphoric acid and the like. Preferred acids for formingacid addition salts include HCl and acetic acid.

The boronate esters of boronic acid compounds of the present inventionare also preferred. These esters are formed by reacting the acid groupsof the boronic acid with a hydroxy compound. Preferred hydroxy compoundsare dihydroxy compounds, especially pinacol, perfluoropinacol,pinanediol, ethylene glycol, diethylene glycol, 1,2-cyclohexanediol,1,3-propanediol, 2,3-butanediol, glycerol or diethanolamine.

The P moiety of the proteasome inhibitor of formula (1a) is preferablyone of R⁷'C(O)—, R⁷—SO₂—, R⁷—NH—C(O)— or R⁷—O—C(O)—, and R⁷ is one ofalkyl, cycloalkyl, aryl, aralkyl, heteroaryl or heteroarylalkyl, thering portion of any of which can be optionally substituted, or if Y isR⁷—C(O)— or R⁷—SO₂—, then R⁷ can also be a saturated or partiallyunsaturated heterocycle.

More preferably, P is one of R⁷—C(O)— or R⁷—SO₂—, and R⁷ is one of aryl,aralkyl, heteroaryl or heteroarylalkyl, any of which can be optionallysubstituted, or a saturated or partially unsaturated heterocycle.

Where R⁷ is alkyl, it is preferably straight chained or branched alkylof from 1 to 6 carbon atoms, more preferably 1–4 carbon atoms. Usefulvalues include methyl, ethyl, propyl, butyl, isopropyl, isobutyl andtert-butyl, with methyl being most preferred. Additionally, where R⁷ isalkaryl, aralkyl or heteroarylalkyl, the alkyl moiety thereof is alsopreferably one having from 1 to 4 carbon atoms, and most preferably 1carbon atom.

Where R⁷ is aryl, it is preferably aryl of from 5 to 10 carbon atoms,more preferably 6 to 10 carbon atoms. Where R⁷ is heteroaryl, one ormore of the carbon atoms of the aforementioned aryl is replaced by oneto three of O, N, or S. The aryl and heteroaryl moieties may, ifdesired, be ring substituted. Useful ring substituents include one ortwo of hydroxy, nitro, trifluoromethyl, halogen, alkyl, alkoxy, cyano,C₆₋₁₀ aryl, benzyl, carboxyalkoxy, amino, and guanidino. Preferredsubstituents include halogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, phenyl andbenzyl. Additionally, where R⁷ is alkaryl, aralkyl or heteroarylalkyl,the above statements equally apply.

Useful R⁷ aryl and aralkyl groups include phenyl, 4-tolyl, benzyl,phenethyl, naphthyl, and naphthylmethyl.

Preferred heteroaryl groups are quinolinyl, quinoxalinyl, pyridyl,pyrazinyl, furanyl or pyrrolyl. Useful values of R⁷ heteroaryl include8-quinolinyl, 2-quinoxalinyl, 2-pyrazinyl, 3-furanyl, 2-pyridyl,3-pyridyl and 4-pyridyl.

Preferred saturated or partially saturated heterocycle moieties are 5-,6-, 9- and 10-membered heterocycles having one, two or three ringheteroatoms selected from O, S or N. A useful value is N-morpholinyl.

Preferred cycloalkyl moieties include C₃₋₁₀ cycloalkyl. Useful valuesinclude cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and cyclononyl.

Especially preferred values of P are 2-pyrazinecarbonyl,8-quinolinesulfonyl and N-morpholinoyl.

As noted above, A in formula (1a) and (1b) can be either 0, 1 or 2.Thus, when A is zero, the residue within the brackets is not present andthe inhibitor is a dipeptide. Similarly, where A is 1, the amino acid orisosteric residue within the brackets is present and the inhibitor is atripeptide. Where A is 2, the inhibitor is a tetrapeptide. Mostpreferably, A is zero.

It is preferred that R¹, R², and R³ in formula (1a) and (1b) are eachindependently one of hydrogen, C₁₋₈ alkyl, C₃₋₁₀ cycloalkyl, C₁₋₆ aryl,a 5-, 6-, 9- or 10-membered heteroaryl group, or —CH₂—R⁵, and morepreferably C₁₋₈ alkyl or —CH₂—R⁵ wherein R¹, R², R³ and R⁵ areoptionally substituted. More preferably, R¹, R² and R³ are eachindependently one of C₁₋₄ alkyl, e.g., methyl, ethyl, propyl, butyl,isopropyl, isobutyl, sec-butyl and t-butyl, or —CH₂—R⁵, where R⁵ is oneof cycloalkyl, aryl or heterocycle. R⁵ is preferably one of C₆₋₁₀ aryl,C₆₋₁₀ ar(C₁₋₆)alkyl, C₁₋₆ alk(C₆₋₁₀)aryl, C₃₋₁₀ cycloalkyl, C₁₋₈ alkoxy,C₁₋₈ alkylthio or a 5-, 6-, 9- or 10-membered heteroaryl group.

The ring portion of any of said aryl, aralkyl, alkaryl or 5-, 6-, 9- or10-membered heteroaryl groups of R¹, R², R³ and R⁵ can be optionallysubstituted by one or two substituents independently selected from thegroup consisting of C₁₋₆ alkyl, C₃₋₈ cycloalkyl, C₁₋₆alkyl(C₃₋₈)cycloalkyl, C₂₋₈ alkenyl, C₂₋₈ alkynyl, cyano, amino, C₁₋₆alkylamino, di(C₁₋₆)alkylamino, benzylamino, dibenzylamino, nitro,carboxy, carbo(C₁₋₆)alkoxy, trifluoromethyl, halogen, C₁₋₆ alkoxy, C₆₋₁₀aryl, C₆₋₁₀ aryl(C₁₋₆)alkyl, C₆₋₁₀ aryl(C₁₋₆)alkoxy, hydroxy, C₁₋₆alkylthio, C₁₋₆ alkylsulfinyl, C₁₋₆ alkylsulfonyl, C₆₋₁₀ arylthio, C₆₋₁₀arylsulfinyl, C₆₋₁₀ arylsulfonyl, C₆₋₁₀ aryl, C₁₋₆ alkyl(C₆₋₁₀)aryl, andhalo(C₆₋₁₀)aryl.

It is more preferred that at least one of R¹ and R² is isobutyl or—CH₂—R⁵, and most preferred that R² is —CH₂—R⁵. It is preferred that R⁵is C₆₋₁₀ aryl, a 5-, 6-, 9- or 10-membered heteroaryl group having oneto three heteroatoms independently selected from O, N and S.

Most preferably, R² is isobutyl, 6-quinolinylmethyl, 3-indolylmethyl,4-pyridylmethyl, 3-pyridylmethyl, 2-pyridylmethyl, benzyl,1-naphthylmethyl, 2-naphthylmethyl, 4-fluorobenzyl, 4-benzyloxybenzyl,4-(2′-pyridylmethoxy)benzyl or benzylnaphthylmethyl.

Preferably, R³ is C₁₋₁₂ alkyl, more preferably C₁₋₆ alkyl, mostpreferably C₄ alkyl, such as isobutyl.

Where R¹, R² or R³ is a substituted alkyl, it is preferably C₁₋₄ alkylsubstituted with at least one cycloalkyl group, preferably a C₅₋₆cycloalkyl group.

Where R¹, R², R³, or R⁵ is substituted aryl or substituted heterocycle,it is preferably substituted with at least one C₁₋₄ alkyl group.

Where R¹, R², R³ or R⁵ is cycloalkyl, it is preferably C₅₋₆ cycloalkyl,e.g., cyclopentyl or cyclohexyl, and can be optionally substituted withat least one C₆₋₁₀ aryl group or at least one alkyl group, preferably aC₁₋₄ alkyl group.

Where R⁵ is —W—R⁶, W is a chalcogen, preferably oxygen or sulfur, morepreferably sulfur, and R⁶ is alkyl, preferably C₁₋₄ alkyl, e.g., methyl,ethyl, propyl, butyl, or isomers thereof.

Preferred values of R include hydrogen or C₁₋₈ alkyl, more preferablyC₁₋₄ alkyl. Useful values of R include methyl, ethyl, isopropyl,isobutyl and n-butyl. Additionally, R can form together with theadjacent R¹, or when A is zero, form together with the adjacent R², anitrogen-containing mono-, bi- or tri-cyclic, saturated or partiallysaturated ring system having 4–14 ring members, and can be optionallysubstituted by one or two of keto, hydroxy, aryl, alkoxy or aryloxy. Itis preferred that the ring system be chosen from one of:

The nitrogen in each of the above formulae is attached to P in formula(1a) and the open valence carbon is attached to either X¹ or X².

It is preferred that Z¹ and Z² are each independently one of C₁₋₄ alkyl,hydroxy, C₁₋₆ alkoxy, and C₆₋₁₀ aryloxy; or together Z¹ and Z²preferably form a moiety derived from a dihydroxy compound selected fromthe group consisting of pinacol, perfluoropinacol, pinanediol, ethyleneglycol, diethylene glycol, 1,2-cyclohexanediol, 1,3-propanediol,2,3-butanediol, glycerol or diethanolamine, or other equivalentsapparent to those skilled in the art. Useful values include methyl,ethyl, propyl and n-butyl. Most preferably, Z¹ and Z² are hydroxy.

A preferred embodiment of the invention is directed to a subgenus ofcompounds having formula (1a) above, where P is R⁷—C(O)— or R⁷—SO₂—, andR⁷ is one of quinolinyl, quinoxalinyl, pyridyl, pyrazinyl, furanyl orpyrrolyl, and when P is R⁷—C(O)—, R⁷ can also be N-morpholinyl.

A preferred group of compounds of this embodiment are compounds offormula (1a) wherein P is one of quinolinecarbonyl, pyridinecarbonyl,quinolinesulfonyl, quinoxalinecarbonyl, quinoxalinesulfonyl,pyrazinecarbonyl, pyrazinesulfonyl, furancarbonyl, furansulfonyl orN-morpholinylcarbonyl; A is zero; X² is —C(O)—NH—; R is hydrogen or C₁₋₈alkyl; R² and R³ are each independently one of hydrogen, C₁₋₈alkyl,C₃₋₁₀cycloalkyl, C₆₋₁₀aryl, C₆₋₁₀ar(C₁₋₆)alkyl, pyridylmethyl, orquinolinylmethyl; and Z¹ and Z² are both hydroxy, C₁₋₆alkoxy, orC₆₋₁₀aryloxy, or together Z¹ and Z² form a moiety derived from adihydroxy compound selected from the group consisting of pinacol,perfluoropinacol, pinanediol, ethylene glycol, diethylene glycol,1,2-cyclohexanediol, 1,3-propanediol, 2,3-butanediol, glycerol ordiethanolamine.

Even more preferred are those compounds wherein: P is8-quinolinecarbonyl, 8-quinolinesulfonyl, 2-quinoxalinecarbonyl,2-quinoxalinesulfonyl, 2-pyrazinecarbonyl; 2-pyrazinesulfonyl,3-pyridinecarbonyl, 3-pyridinesulfonyl, 3-furancarbonyl, 3-furansulfonylor N-morpholinecarbonyl; R is hydrogen; R³ is isobutyl; R² is isobutyl,1-naphthylmethyl, 2-naphthylmethyl, 3-pyridylmethyl, 2-pyridylmethyl6-quinolinylmethyl, 3-indolylmethyl, benzyl, 4-fluorobenzyl,4-hydroxybenzyl, 4-(2′-pyridylmethoxy)benzyl, 4-(benzyloxy)benzyl,benzylnaphthylmethyl or phenethyl; and Z¹ and Z² are both hydroxy, ortogether Z¹ and Z² form a moiety derived from a dihydroxy compoundselected from the group consisting of pinacol, perfluoropinacol,pinanediol, ethylene glycol, diethylene glycol, 1,2-cyclohexanediol,1,3-propanediol, 2,3-butanediol, glycerol or diethanolamine.

Another preferred embodiment of the present invention is directed tocompounds of formula (1a) where A is zero. These compounds possessunexpectedly high potency and selectivity as inhibitors of proteasomefunction.

A third preferred subgenus of compounds are compounds of formula (1a)where one of R¹, R² or R³ corresponds to an amino acid side-chaincorresponding to tyrosine or an O-substituted tyrosine derivative,formed by reacting the hydroxyl group of the tyrosine side-chain with acompound having a reactive functional group. This subgenus includescompounds having the formula (1a), wherein at least one R¹, R² or R³ is:

where R⁹ is one of hydrogen, alkyl, cycloalkyl, aryl, aralkyl,heteroaryl or heteroarylalkyl, wherein the alkyl is optionallysubstituted with one of C₁₋₆ alkyl, halogen, monohalo (C₁₋₆) alkyl, andtrifluoromethyl; and wherein said cycloalkyl, aryl, aralkyl, heteroaryland heteroarylalkyl groups can be optionally substituted with one or twoof C₁₋₆ alkyl, C₃₋₈ cycloalkyl, C₁₋₆ alkyl(C₃₋₈)cycloalkyl, C₂₋₈alkenyl, C₂₋₈ alkynyl, cyano, amino, C₁₋₆ alkylamino,di(C₁₋₆)alkylamino, benzylamino, dibenzylamino, nitro, carboxy,carbo(C₁₋₆)alkoxy, trifluoromethyl, halogen, C₁₋₆alkoxy, C₆₋₁₀ryl,C₆₋₁₀aryl(C₁₋₆)alkyl, C₆₋₁₀aryl(C₁₋₆)alkoxy, hydroxy, C₁₋₆alkylthio,C₁₋₆alkylsulfinyl, C₁₋₆alkylsulfonyl, C₆₋₁₀arylthio, C₆₋₁₀arylsulfinyl,C₆₋₁₀arylsulfonyl, C₆₋₁₀aryl, C₁₋₆alkyl(C₆₋₁₀)aryl, and halo(C₆₋₁₀)aryl;and A¹ and A 2 are independently one of hydrogen, C₁₋₆alkyl, halogen,monohalo(C₁₋₆)alkyl, or trifluoromethyl.

The group —O—R⁹ is in either the ortho- or para-position, with para-being preferred. The groups A¹ and A² can be at any remaining positionson the phenyl ring.

It is preferred that R⁹ is one of C₁₋₈alkyl, C₃₋₁₀cycloalkyl, C₆₋₁₀aryl,C₆₋₁₀ar(C₁₋₆)alkyl, 5- to 10-membered heteroaryl or 5- to 10-memberedheteroaryl(C₁₋₆)alkyl.

Useful values of R⁹ include benzyl, phenethyl, pyridyl, pyridylmethyl,furanylmethyl pyrrolymethyl, pyrrolidylmethyl, oxazolylmethyl andimidazolylmethyl.

The ring portion of any of said aryl, aralkyl, alkaryl or 5-, 6-, 9- or10-membered heteroaryl groups of R¹, R², R³ and R⁵ can be optionallysubstituted by one or two substituents independently selected from thegroup consisting of C₁₋₆ alkyl, C₃₋₈ cycloalkyl, C₁₋₆alkyl(C₃₋₈)cycloalkyl, C₂₋₈ alkenyl, C₂₋₈ alkynyl, cyano, amino, C₁₋₆alkylamino, di(C₁₋₆)alkylamino, benzylamino, dibenzylamino, nitro,carboxy, carbo(C₁₋₆)alkoxy, trifluoromethyl, halogen, C₁₋₆ alkoxy, C₆₋₁₀aryl, C₆₋₁₀ aryl(C₁₋₆)alkyl, C₆₋₁₀ aryl(C₁₋₆)alkoxy, hydroxy, C₁₋₆alkylthio, C₁₋₆ alkylsulfinyl, C₁₋₆ alkylsulfonyl, C₆₋₁₀ arylthio, C₆₋₁₀arylsulfinyl, C₆₋₁₀ arylsulfonyl, C₆₋₁₀ aryl, C₁₋₆ alkyl(C₆₋₁₀)aryl, andhalo(C₆₋₁₀)aryl.

A preferred class of compounds of this embodiment are compounds offormula (1a) wherein: A is zero; P is one of R⁷—C(O)—, R⁷—SO₂—,R⁷—NH—C(O)— or R⁷—O—C(O)—; R⁷ is one of quinolinyl, quinoxalinyl,pyridyl, pyrazinyl, furanyl or pyrrolyl, or when P is R⁷—C(O)—, R⁷ canalso be N-morpholinyl; X² is —C(O)—NH—; R³ is C₁₋₆alkyl; R² is:

where A¹ and A² are independently one of hydrogen, C₁₋₆ alkyl, halogen,monohalo(C₁₋₆)alkyl or trifluoromethyl; and R⁹ is one of hydrogen, C₁₋₆alkyl, phenyl, benzyl, phenethyl or pyridylmethyl; and

Z¹ and Z² are both hydroxy, C₁₋₆alkoxy, or C₆₋₁₀aryloxy, or together Z¹and Z² form a moiety derived from a dihydroxy compound selected from thegroup consisting of pinacol, perfluoropinacol, pinanediol, ethyleneglycol, diethylene glycol, 1,2-cyclohexanediol, 1,3-propanediol,2,3-butanediol, glycerol or diethanolamine.

Even more preferred are compounds of formula (1a) wherein: A is zero; Pis 8-quinolinecarbonyl, 8-quinolinesulfonyl, 2-quinoxalinecarbonyl,2-quinoxalinesulfonyl, 2-pyrazinecarbonyl, 2-pyrazinesulfonyl,3-pyridinecarbonyl, 3-pyridinesulfonyl, 3-furancarbonyl, 3-furansulfonylor N-morpholinecarbonyl; X² is —C(O)—NH—; R³ is isobutyl; R² is:

where A¹ and A² are independently one of hydrogen, methyl, ethyl,chloro, fluoro, or trifluoromethyl; and R⁹ is one of hydrogen, methyl,ethyl, butyl, phenyl, benzyl, phenethyl or pyridylmethyl; and

Z¹ and Z² are both hydroxy, or together Z¹ and Z² form a moiety derivedfrom a dihydroxy compound selected from the group consisting of pinacol,perfluoropinacol, pinanediol, ethylene glycol, diethylene glycol,1,2-cyclohexanediol, 1,3-propanediol, 2,3-butanediol, glycerol ordiethanolamine.

A fourth preferred subgenus of compounds includes compounds of formula(1a) wherein one of the amino acid side-chains, preferably theside-chain defined by R², is an unnatural amino acid selected fromnaphthylmethyl, pyridylmethyl and quinolinylmethyl, withquinolinylmethyl being most preferred. Thus, this subgenus includescompounds of formula (1a), wherein at least one R¹, R² or R³ isnaphthylmethyl, pyridylmethyl or quinolinylmethyl; provided that thecompound is other thanisovaleryl-phenylalanine-norvaline-[(naphthylmethyl),(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)]methylamide or(3-t-butylsulfonyl)propionyl-norvaline-(1-naphthyl,dihydroxyboryl)methylamide.

A fifth preferred subgenus includes compounds of formula (1a) where R,together with R¹, or with R² when A is zero, forms a nitrogen containingheterocycle. This subgenus includes compounds having formula (1a),wherein:

R forms together with the adjacent R¹, or when A is zero, forms togetherwith the adjacent R², a nitrogen-containing mono-, bi- or tri-cyclic,saturated or partially saturated ring system having 4–14 ring members,and one or two optional substituents selected from the group consistingof keto, hydroxy, aryl, alkoxy and aryloxy;

when A is 2, the R¹ that is not adjacent to N—R is one of hydrogen,alkyl, cycloalkyl, aryl, heterocycle or —CH₂—R⁵; and when A is 1 or 2,R² is one of hydrogen, alkyl, cycloalkyl, aryl, heterocycle or —CH₂—R⁵,where R⁵ is defined as above.

A preferred class of compounds of this embodiment of the invention arethose wherein: A is zero; P is hydrogen; X² is —C(O)—NH—; and R formstogether with the adjacent R², one of the nitrogen-containing ringsystems shown in the above structures; R³ is C₁₋₆alkyl; and Z¹ and Z²are both hydroxy, C₁₋₆alkoxy, or C₆₋₁₀aryloxy, or together Z¹ and Z²form a moiety derived from a dihydroxy compound selected from the groupconsisting of pinacol, perfluoropinacol, pinanediol, ethylene glycol,diethylene glycol, 1,2-cyclohexanediol, 1,3-propanediol, 2,3-butanediol,glycerol or diethanolamine. The hydrochloride salts of these compoundsare also especially preferred.

Even more preferred are those compounds wherein R forms together withthe adjacent R², a nitrogen-containing ring system having one of thestructures shown above; R³ is isobutyl; and Z¹ and Z² are both hydroxy,or together Z¹ and Z² form a moiety derived from a dihydroxy compoundselected from the group consisting of pinacol, perfluoropinacol,pinanediol, ethylene glycol, diethylene glycol, 1,2-cyclohexanediol,1,3-propanediol, 2,3-butanediol, glycerol or diethanolamine.

Examples of suitable proteasome inhibitors include without limitationthe following compounds, as well as pharmaceutically acceptable saltsand boronate esters thereof:

-   N-(4-morpholine)carbonyl-β-(1-naphthyl)-L-alanine-L-leucine boronic    acid,-   N-(8-quinoline)sulfonyl-β-(1-naphthyl)-L-alanine-L-leucine boronic    acid,-   N-(2-pyrazine)carbonyl-L-phenylalanine-L-leucine boronic acid,-   L-proline-L-leucine boronic acid,-   N-(2-quinoline)carbonyl-L-homophenylalanine-L-leucine boronic acid,-   N-(3-pyridine)carbonyl-L-phenylalanine-L-leucine boronic acid,-   N-(3-phenylpropionyl)-L-phenylalanine-L-leucine boronic acid,-   N-(4-morpholine)carbonyl-L-phenylalanine-L-leucine boronic acid,-   N-(4-morpholine)carbonyl-(O-benzyl)-L-tyrosine-L-leucine boronic    acid,-   N-(4-morpholine)carbonyl-L-tyrosine-L-leucine boronic acid, and-   N-(4-morpholine)carbonyl-[O-(2-pyridylmethyl)]-L-tyrosine-L-leucine    boronic acid.

Preferred compounds having formula (2a) include compounds where Y is oneof R⁸—C(O)—, R⁸—SO₂—, R⁸—NH—C(O)— or R⁸—O—C(O)—, and

R⁸ is one of C₆₋₁₀ aryl, C₆₋₁₀ ar(C₁₋₆)alkyl, or a 5–10 memberedheteroaryl, any of which can be optionally substituted, or when P isR⁸—C(O)—, R⁸ can also be N-morpholinyl; provided that when Y isR⁸—C(O)—, then R⁸ is other than phenyl, benzyl or C₁₋₃ alkyl.

Where R⁸ is alkyl, it is preferably alkyl of from 1 to 4 carbon atoms,e.g., methyl, ethyl, propyl, butyl, or isomers thereof. Additionally,where R⁸ is alkaryl or aralkyl, the alkyl moiety thereof is alsopreferably one having from 1 to 4 carbon atoms.

Where R⁸ is aryl, it is preferably aryl of from 6 to 10 carbon atoms,e.g., phenyl or naphthyl, which may, if desired, be ring substituted.Additionally, where R⁸ is alkaryl, aralkyl, aryloxy, alkaryloxy, oraralkoxy, the aryl moiety thereof is also preferably one having from 5to 10 carbon atoms, most preferably 6 to 10 carbon atoms. Preferably,the R⁸ moiety is a saturated, partially unsaturated or aromaticheterocycle, more preferably an isomeric pyridine ring or morpholinering.

Y is most preferably one of:

where R⁴ is C₆₋₁₂ alkyl.

In an additional preferred embodiment of the present invention, the Ymoiety of the proteasome inhibitor of formula (2a) is an isosteric aminoacid replacement of formula (3a):

where R¹ is as defined for formula (1a) above. Useful and preferredvalues of R¹ are the same as those defined for formula (1a) above; and

P is one of R⁷—C(O)—, R⁷—SO₂—, R⁷—NH—C(O)— or R⁷—O—C(O)—, and R⁷ is oneof alkyl, aryl, alkaryl, aralkyl, any of which can be optionallysubstituted, or when Y is R⁷—C(O)— or R⁷—SO₂—, R⁷ can also be anoptionally substituted 5–10 membered saturated, partially unsaturated oraromatic heterocycle.

Useful and preferred values of R⁷, when R⁷ is one of alkyl, aryl,alkaryl, aralkyl, any of which are optionally substituted are as definedfor formula (1a) above. When R⁷ is optionally substituted 5–10 memberedsaturated, partially unsaturated or aromatic heterocycle, preferred anduseful values are as defined for heteroaryl, unsaturated and partiallysaturated heterocycle of the R⁷ of formula (1a). In this aspect of theinvention Y is most preferably:

In either embodiment of the compounds of formula (2a), useful andpreferred values of R³ are the same as for formula (1a) above.

In formula (1a) and (1b), X¹ represents a peptide bond or an isosterethat can be used as a peptide bond replacement in the proteasomeinhibitors to increase bioavailability and reduce hydrolytic metabolism.As noted above, X¹ can be one of —C(O)NH—, —CH₂—NH—, —CH(OH)—CH(OH)—,—CH(OH)—CH₂—CH(OH)—CH₂—NH—, —CH═CH—, —C(O)—CH₂—, —SO₂—NH—, —SO₂—CH₂— or—CH(OH)—CH₂—C(O)—NH—. Preferably, X¹ is —C(O)—NH—.

Introduction of these X¹ moieties into the proteasome inhibitors resultsin the following wherein R_(x) and R_(y) have the same definitions as R¹and R², above and P, Z¹, Z² and R³ are defined as above for formula(1a).

Thus, for example, if Z-Leu-Leu-Leu-B(OH)₂ is found to undergo rapidhydrolytic metabolism to produce Z-Leu-OH and H₂-N-Leu-Leu-B(OH)₂, thehydroxyethylene isostere can be prepared to eliminate this reaction:

Another group of compounds of the present invention are aza-peptideisosteres. This is the result of the replacement of the α-carbon atom ofan amino acid with a nitrogen atom, e.g.,

wherein R_(x) represents R¹, R_(y) represents R², P, Z¹, Z² and R³ aredefined as above for formula (1a) and (1b).

When P and R are both H, formula (1) will exist in equilibrium with acyclic formula (4), which is considered to be covered by the currentinvention:

The above-described boronic ester and acid compounds include both D andL peptidyl configurations. However, L configurations are preferred.

The present invention relates to a method for reducing the rate ofmuscle protein degradation in a cell comprising contacting the cell witha proteasome inhibitor described above. More specifically, the presentinvention relates to a method for reducing the rate of loss of musclemass in an animal comprising contacting cells of the muscle with aproteasome inhibitor described above.

The present invention also relates to a method for reducing the activityof NF-κB in a cell comprising contacting the cell with a proteasomeinhibitor described above. More specifically, the present invention alsorelates to a method for reducing the activity of NF-κB in an animalcomprising contacting cells of the animal with a proteasome inhibitordescribed above.

The present invention also relates to a method for reducing the rate ofproteasome-dependent intracellular protein breakdown comprisingcontacting cells with a proteasome inhibitor described above. Morespecifically, the present invention also relates to a method forreducing the rate of intracellular protein breakdown in an animalcomprising contacting cells of the animal with the proteasome inhibitordescribed above.

The present invention further relates to a method of reducing the rateof degradation of p53 protein in a cell comprising administering to thecell a proteasome inhibitor described above More specifically, thepresent invention further provides a method of reducing the rate ofdegradation of p53 protein in an animal (preferably, an animal subjectedto DNA damaging drugs or radiation) comprising administering to saidanimal a proteasome inhibitor described above.

The present invention further relates to a method for inhibiting cyclindegradation in a cell comprising contacting said cells with a proteasomeinhibitor described above. More specifically, the present inventionrelates to a method for inhibiting cyclin degradation in an animalcomprising contacting cells of said animal with a proteasome inhibitordescribed above.

The present invention also provides a method for treating cancer,psoriasis, restenosis, or other cell proliferative diseases in a patientcomprising administering to the patient a proteasome inhibitor describedabove.

The present invention also relates to a method for inhibiting antigenpresentation in a cell comprising administering to the cell a proteasomeinhibitor described above. More specifically, the present inventionrelates to a method for inhibiting antigen presentation in animalcomprising administering to the animal a proteasome inhibitor describedabove.

The present invention further provides a method for inhibiting inducibleNF-κB dependent cell adhesion in an animal comprising administering tosaid animal a proteasome inhibitor described above.

The present invention also provides a method for inhibiting HIVinfection in an animal comprising administering to said animal aproteasome inhibitor described above.

The “animals” referred to herein are preferably mammals. Both terms areintended to include humans.

Preferably, the methods described above deliver the proteasome inhibitorby either contacting cells of the animal with a proteasome inhibitordescribed above or by administering to the animal a proteasome inhibitordescribed above.

The compounds of the present invention inhibit the functioning of theproteasome. This proteasome-inhibition activity results in theinhibition or blocking of a variety of intracellular functions. Inparticular, inhibition of proteasome function inhibits the activation orprocessing of transcription factor NF-κB. NF-κB plays a central role inthe regulation of a diverse set of genes involved in the immune andinflammatory responses. Inhibition of proteasome function also inhibitthe ubiquitination/proteolysis pathway. This pathway catalyzes selectivedegradation of highly abnormal proteins and short-lived regulatoryproteins. The ubiquitination proteolysis pathway also is involved in theprocessing of internalized cellular or viral antigens into antigenicpeptides that bind to MHC-I molecules. Thus, the proteasome inhibitorsof the present invention can be used in reducing the activity of thecytosolic ATP-ubiquitin-dependent proteolytic system in a number of celltypes.

The inhibitors can be used in vitro or in vivo. They can be administeredby any number of known routes, including orally, intravenously,intramuscularly, subcutaneously, intrathecally, topically, and byinfusion (Platt et al., U.S. Pat. No. 4,510,130; Badalamente et al.,Proc. Natl. Acad. Sci U.S.A. 86:5983–5987 (1989); Staubli et al., BrainResearch 444:153–158 (1988)) and will generally be administered incombination with a physiologically acceptable carrier (e.g.,physiological saline). The effective quantity of inhibitor given will bedetermined empirically and will be based on such considerations as theparticular inhibitor used, the condition of the individual, and the sizeand weight of the individual. It is to be expected that the generalend-use application dose range will be about 0.01 to 100 mg per kg perday, preferably 0.1 to 75 mg per kg per day for an effective therapeuticeffect.

The present invention relates to a method of inhibiting (reducing orpreventing) the accelerated or enhanced proteolysis that occurs inatrophying muscles and is known to be due to activation of anonlysosomal ATP-requiring process in which ubiquitin plays a criticalrole.

Inhibition of the ATP-ubiquitin-dependent pathway is a new approach fortreating the negative nitrogen balance in catabolic states. This can beeffected through use of an inhibitor of the present invention, resultingin reduction of loss of muscle mass in conditions in which it occurs.Excessive protein loss is common in many types of patients, includingindividuals with sepsis, burns, trauma, many cancers, chronic orsystemic infections, neuromotor degenerative disease, such as musculardystrophy, acidosis, or spinal or nerve injuries. It also occurs inindividuals receiving corticosteroids, and those in whom food intake isreduced and/or absorption is compromised. Moreover, inhibitors of theprotein breakdown pathway could possibly be valuable in animals, e.g.,for combating “shipping fever”, which often leads to a major weight lossin cattle or pigs.

The accelerated proteolysis evident in atrophy of skeletal muscles upondenervation or fasting is catalyzed by the nonlysosomal ATP-dependentdegradative pathway. It has been shown that in a variety of catabolicstates (e.g., denervation, fasting, fever, certain endocrinopathies ormetabolic acidosis) muscle wasting is due primarily to acceleratedprotein breakdown and, in addition, that the increased proteolysisresults from activation of the cytosolic ATP-ubiquitin-dependentproteolytic system, which previously had been believed to serve only inthe rapid elimination of abnormal proteins and certain short-livedenzymes. The discovery that this pathway is responsible for theaccelerated proteolysis in these catabolic states is based on studies inwhich different proteolytic pathways were blocked or measuredselectively in incubated muscles, and the finding of increased mRNA forcomponents of this pathway (e.g., for ubiquitin and proteasome subunits)and increased levels of ubiquitin-protein conjugates in the atrophyingmuscles. The nonlysosomal ATP-ubiquitin-dependent proteolytic processincreases in muscle in these conditions and is responsible for most ofthe accelerated proteolysis that occurs in atrophying muscles. There isa specific increase in ubiquitin mRNA, induction of mRNA for proteasomeand increased ubiquitinated protein content in atrophying muscles thatis not seen in non-muscle tissue under the same conditions.

The inhibitors of the present invention can be used to reduce (totallyor partially) the nonlysosomal ATP-dependent protein degradation shownto be responsible for most of the increased protein degradation thatoccurs during fasting, denervation, or disuse (inactivity), steroidtherapy, febrile infection, and other conditions.

One approach to testing drug candidates for their ability to inhibit theATP-ubiquitin-dependent degradative process is to measure proteolysis incultured cells (Rock, et al., Cell 78:761 (1994)). For example, thedegradation of long-lived intracellular proteins can be measured inmouse C2C12 myoblast cells. Cells are incubated with ³⁵S-methionine for48 hours to label long-lived proteins and then chased for 2 hours withmedium containing unlabeled methionine. After the chase period, thecells are incubated for 4 hours in the presence or absence of the testcompound. The amount of protein degradation in the cell can be measuredby quantitating the trichloroacetic acid soluble radioactivity releasedfrom the pre-labeled proteins into the growth medium (an indicator ofintracellular proteolysis).

Inhibitors can also be tested for their ability to reduce muscle wastingin vivo. Urinary excretion of the modified amino acid 3-methyl histidine(3-MH) is probably the most well characterized method for studyingmyofibrillar protein degradation in vivo (see Young and Munro,Federation Proc. 37:229–2300 (1978)). 3-Methylhistidine is apost-translationally modified amino acid which cannot be reutilized forprotein synthesis, and it is only known to occur in actin and myosin. Itoccurs in actin isolated from all sources, including cytoplasmic actinfrom many different cell types. It also occurs in the myosin heavy chainof fast-twitch (white, type II) muscle fibers, but it is absent frommyosin of cardiac muscle and myosin of slow-twitch (red, type I) musclefibers. Due to its presence in actin of other tissues than skeletalmuscle, other tissues will contribute to urinary 3-MH. Skeletal musclehas been estimated to contribute 38–74% of the urinary 3-MH in normalrats and 79–86% of the urinary 3-MH in rats treated with corticosterone(100 mg/kg/day subcutaneously) for 2–4 days (Millward and Bates,Biochem. J. 214:607–615 (1983); Kayali, et al., Am. J. Physiol.252:E621–E626 (1987)).

High-dose glucocorticoid treatment is used to induce a state of musclewasting in rats. Treating rats with daily subcutaneous injections ofcorticosterone (100 mg/kg) causes an increase of approximately 2-fold inurinary 3-MH. The increase in excretion of 3-MH is transient, with apeak increase after 2–4 days of treatment and a return to basal valuesafter 6–7 days of treatment (Odedra, et al., Biochem. J. 214:617–627(1983); Kayali, et al., Am. J. Physiol. 252:E621–E626 (1987)).Glucocorticoids have been shown to activate the ATP-ubiquitin-dependentprotcolytic pathway in skeletal muscle (Wing and Goldberg, Am. J.Physiol. 264:E668–E676 (1993)) and proteasome inhibitors are thereforeexpected to inhibit the muscle wasting that occurs after glucocorticoidtreatment.

The proteasome inhibitors can be administered alone or in combinationwith another inhibitor or an inhibitor of another pathway (e.g., alysosomal or Ca⁺⁺-dependent pathway) responsible for loss of musclemass.

Use of Proteasome Inhibitors as Agents That Selectively Protect NormalCells from DNA Damage During Radiation and Chemotherapy Treatment ofTumors

The inhibitors of the present invention will block the degradation ofthe tumor suppressor protein p53. This protein is degraded by the ATPubiquitin dependent proteolysis by the proteasome (see Scheffner et al.,Cell 75:495–505 (1993)).

Studies of p53 knockout mice indicate an important role for p53 inreducing incidence of tumors (Donehower et al., Nature 356:215–221(1992)). In normal cells expressing wild type, unmutated p53, the basallevels of p53 are very low due to very rapid degradation of p53 protein.However, expression of p53 protein in normal cells is stimulated inresponse to radiation and drugs that induce DNA damage (Kastan et al.,Cancer Res. 51:6304–6311 (1991)). These induced high levels of wildtype, unmutated p53 induce arrest of normal cell proliferation at the G1stage of the cell cycle (Kastan et al., supra; Kuerbitz, PNAS89:7491–7495 (1992)). This arrest of cell proliferation permits repairof damaged DNA. By contrast, in tumor cells expressing mutant forms ofp53, DNA damaging drugs or radiation do not induce cell cycle arrest(Kastan et al., supra; Kastan et al., Cell 71:587–597 (1992)).Consequently, tumor cells are selectively damaged by radiation andcytotoxic drugs.

The selective arrest response of normal cells by inducing p53 suggeststhat enhancing the p53 response can allow the treatment of the tumorwith higher/more prolonged tumoricidal doses of radiation orantineoplastic drugs. The idea that induction of p53 by a non toxicagent as an adjunct to radiotherapy has been reported previously (Lane,Nature 358:15–16 (1992), but a method for reducing it to practice wasnot described.

The use of proteasome inhibitors provides a method for augmenting theexpression of p53 in normal cells by preventing its degradation by theproteasome. An example of this would be the systemic administration ofproteasome inhibitor at a sufficient dose to inhibit p53 degradation bythe proteasome during the treatment of the tumor with cytotoxic drugs orradiation. This will prolong and increase the levels of p53 expressionin normal cells and will enhance the arrest of normal cellproliferation, reducing their sensitivity to higher doses of radiationor cytotoxic drugs. Administration of proteasome inhibitors wouldtherefore permit exposing the tumor to higher doses of radiation,enhancing the killing of tumor cells. Thus, proteasome inhibitors can beused as adjuvants to therapy with tumoricidal agents, such as radiationand cytotoxic drugs.

Topical Application of Proteasome Inhibitors to Enhance p53 Expressionin Skin

The expression of p53 in normal skin is induced by exposure of the skillto UV irradiation, which inhibits DNA replication that is needed forcell division (Maltzman et al., Mol. Cell. Biol. 4:1689 (1984); Hall etal., Oncogene 8:203–207 (1993)). This protects normal skin fromchromosomal DNA damage by allowing time for DNA repair before DNAreplication.

Defects in the p53 response pathway, such as seen with AtaxiaTelangiectasia, result in increased susceptibility to ionizingradiation-induced skin tumors (Kastan et al., Cell 71:587–597 (1992)).It is well established that exposure of normal individuals increases therisk for many kinds of skin cancers. This risk can be diminished by UVfiltering chemicals in skin creams. Another approach would be to promotethe resistance of the DNA in skin cells to UV damage by the topicalapplication of agents that enhance the skin's expression of p53 inresponse to UV light. Inhibiting p53 degradation by the topicalapplication of proteasome inhibitors provides a method to enhance thep53 response.

One preferred embodiment of the present invention is the topicalapplication of proteasome inhibitors to reduce the acknowledged risk ofskin cancers that results from the treatment of psoriasis using UVlight, which is often combined with psoralens or coal tar. Each of theseagents can induce DNA damage.

Use of Proteasome Inhibitors to Reduce the Activity of NF-κB

NF-κB exists in an inactive form in the cytoplasm complexed with aninhibitor protein IκB. In order for the NF-κB to become active andperform its function, it must enter the cell nucleus. It cannot do this,however, until the IκB portion of the complex is removed, a processreferred to by those skilled in the art as the activation of, orprocessing of, NF-κB. In some diseases, the normal performance of itsfunction by the NF-κB can be detrimental to the health of the patient.For example, as mentioned above, NF-κB is essential for the expressionof the human immunodeficiency virus (HIV). Accordingly, a process thatwould prevent the activation of the NF-κB in patients suffering fromsuch diseases could be therapeutically beneficial. The inhibitorsemployed in the practice of the present invention are capable ofpreventing this activation. Thus, blocking NF-κB activity could haveimportant application in various areas of medicine, e.g., inflammation,through the inhibition of expression of inflammatory cytokines and celladhesion molecules, (ref. Grilli et al., International Review ofCytology 143: 1–62 (1993)) sepsis, AIDS, and the like.

More specifically, the activity of NF-κB is highly regulated (Grilli etal., International Review of Cytology 143: 1–62 (1993); Beg et al.,Genes and Development 7:2064–2070 (1993)). NF-κB comprises two subunits,p50 and an additional member of the rel gene family, e.g., p65 (alsoknown as Rel A). In most cells, the p50 and p65 are present in aninactive precursor form in the cytoplasm, bound to IκB. In addition, thep50 subunit of NF-κB is generated by the proteolytic processing of a 105kD precursor protein NF-κB₁ (p105), and this processing is alsoregulated. The sequence of the N-terminal 50 kD portion of p105 issimilar to that of p65 and other members of the rel gene family (the relhomology domain). By contrast, the C-terminal 55 kD of p105 bears astriking resemblance to IκB-α (also known as MAD3). Significantly,unprocessed p105 can associate with p65 and other members of the relfamily to form a p65/p105 heterodimer. Processing of p105 results in theproduction of p50, which can form the transcriptionally active p50/p65heterodimer. The C-terminal IκB-α-homologous sequence of p105 is rapidlydegraded upon processing.

There is another rel-related protein, NF-κB₂ (p100), that is similar top105 in that it, too, is processed to a DNA binding subunit, p52 (Neriet al., Cell 67:1075 (1991); Schmid et al., Nature 352:733 (1991); Bourset al., Molecular and Cellular Biology 12:685 (1992); Mercurio et al.,DNA Cell Biology 11:523 (1992)). Many of the structural and regulatoryfeatures of p100 are similar to p105. In addition, the p100 protein canalso form a heterodimer with p65 and other rel family members.

In summary, the transcriptional activity of heterodimers consisting ofp50 and one of the many rel family proteins, such as p65, can beregulated by at least two mechanisms. First, the heterodimers associatewith IκB-α to form an inactive ternary cytoplasmic complex. Second, therel family members associate with p105 and p100 to form inactivecomplexes. The ternary complex can be activated by the dissociation anddestruction of IκB-α, while the p65/p105 and p65/p100 heterodimer can beactivated by processing p105 and p100, respectively.

The dissociation of IκB-α can be induced by a remarkably large number ofextracellular signals, such as lipopolysaccharides, phorbol esters,TNF-α, and a variety of cytokines. The IκB-α is then rapidly degraded.Recent studies suggest that p105 and p100 processing can also be inducedby at least some of these extracellular signals.

Studies have demonstrated that p105 or a truncated form of p105 (p60Tth)can be processed to p50 in vitro (Fan et al., Nature 354:395–398(1991)). Certain of the requirements and characteristics of this invitro processing reaction (e.g., ATP/Mg⁺⁺ dependency) implicated theinvolvement of the ubiquitin-mediated protein degradation pathway(Goldberg, Eur. J. Biochem. 203:9–23 (1992), Hershko et al., Annu. Rev.Biochem. 61:761–807 (1992)).

The proteasome is required for the processing of p105 to p50.p105/p60Tth proteins are not processed in mammalian cell cytoplasmicextracts depleted of proteasome activity. However, addition of purified26S proteasomes to these depleted extracts restores the processingactivity. Additionally, specific inhibitors of the proteasome block theformation of p50 in mammalian cell extracts and in vivo. Also, mammalianp105 is processed to p50 in Saccharomyces cerevisiae in vivo, and amutant deficient in the chymotrypsin-like activity of the proteasomeshowed a significant decrease in p105 processing. p60Tth isubiquitinated in vitro and this ubiquitination is a pre-requisite forp105 processing.

As mentioned above, the C-terminal half of the p105 (p105C′) is rapidlydegraded during the formation of p50 and the sequence of p105C′ isremarkably similar to that of IκB. IκB-α is rapidly degraded in responseto NF-κB inducers and this degradation has been shown to be necessaryfor the activation (Mellits et al., Nucleic Acids Research21(22):5059–5066 (1993); Henkel et al., Nature 365:182–185 (1993); Beget al., Molecular and Cellular Biology 13(6):3301–3310 (1993)). IκB-αdegradation and the activation of NF-κB are also blocked by inhibitorsof proteasome function or ubiquitin conjugation (Palombella et al., Cell78:773–785 (1994)).

Accordingly, the proteasome plays an essential role in the regulation ofNF-κB activity. First, the proteasome is required for the processing ofp105 and possibly p100. The degradation of the inhibitory C-terminus canalso require the proteasome. Second, the proteasome appears to berequired for the degradation of IκB-α in response to extracellularinducers.

The present invention relates to a method for reducing the activity ofNF-κB in an animal comprising contacting cells of the animal withinhibitors of proteasome function.

Compounds can be tested for their ability to inhibit the activation ofNF-κB by means of a DNA binding assay (Palombella, et al., Cell 78:773(1994)). Whole-cell extracts are prepared from untreated or TNF-αtreated cells that have been pretreated for 1 hour with the testcompound. The DNA binding activity of NF-κB is measured by anelectrophoretic mobility shift assay using the PRDII probe from thehuman IFN-β gene promoter.

As an indirect measure of NF-κB activation, the cell-surface expressionof E-selectin, I-CAM-1, and V-CAM-1 on primary human umbilical veinendothelial cells (HUVECs) can be determined by means of a cell surfacefluorescent immuno-binding assay. Because E-selectin, I-CAM-1, andV-CAM-1 are under the regulatory control of NF-κB, inhibition of NF-κBactivation results in reduced levels of these adhesion molecules on thecell surface.

Compounds can also be tested for their ability to inhibit a delayed-typehypersensitivity response in mice. Contact hypersensitivity is amanifestation of an in vivo T-cell mediated immune response (Friedmann,Curr. Opinion Immunology, 1:690–693 (1989)). Although the exactmolecular mechanisms that regulate the cellular interactions andvascular changes involved in the response remain obscure, it is clearthat the process is dependent upon the interplay of soluble mediators,adhesion molecules, and the cytokine network (Piguet, et al., J. Exp.Med. 173:673–679 (1991); Nickoloff, et al., J. Invest. Dermatol.94:151S–157S (1990)). NF-κB, by mediating events such as the productionof cytokines and the induction and utilization of cell-surface adhesionmolecules, is a central and coordinating regulator involved in immuneresponses.

The compounds of formula (1b) or (2b) can be used to treat chronic oracute inflammation that is the result of transplantation rejection,arthritis, rheumatoid arthritis, infection, dermatosis, inflammatorybowel disease, asthma, osteoporosis, osteoarthritis and autoimmunedisease. Additionally, inflammation associated with psoriasis andrestenosis can also be treated.

The term “treatment of inflammation” or “treating inflammation” isintended to include the administration of compounds of the presentinvention to a subject for purposes which can include prophylaxis,amelioration, prevention or cure of an inflammatory response. Suchtreatment need not necessarily completely ameliorate the inflammatoryresponse. Further, such treatment can be used in conjunction with othertraditional treatments for reducing the inflammatory condition known tothose of skill in the art.

The proteasome inhibitors of the invention can be provided as a“preventive” treatment before detection of an inflammatory state, so asto prevent the same from developing in patients at high risk for thesame, such as, for example, transplant patients.

In another embodiment, efficacious levels of the proteasome inhibitorsof the invention are administered so as to provide therapeutic benefitsagainst the secondary harmful inflammatory effects of inflammation. Byan “efficacious level” of a composition of the invention is meant alevel at which some relief is afforded to the patient who is therecipient of the treatment. By an “abnormal” host inflammatory conditionis meant an level of inflammation in the subject at a site which exceedsthe norm for the healthy medical state of the subject, or exceeds adesired level. By “secondary” tissue damage or toxic effects is meantthe tissue damage or toxic effects which occur to otherwise healthytissues, organs, and the cells therein, due to the presence of aninflammatory response, including as a result of a “primary” inflammatoryresponse elsewhere in the body.

Amounts and regimens for the administration of proteasome inhibitors andcompositions of the invention can be determined readily by those withordinary skill in the clinical art of treating inflammation-relateddisorders such as arthritis, tissue injury and tissue rejection.Generally, the dosage of the composition of the invention will varydepending upon considerations such as: type of pharmaceuticalcomposition employed; age; health; medical conditions being treated;kind of concurrent treatment, if any, frequency of treatment and thenature of the effect desired; extent of tissue damage; gender; durationof the symptoms; and, counter indications, if any, and other variablesto be adjusted by the individual physician. A desired dosage can beadministered in one or more applications to obtain the desired results.Pharmaceutical compositions containing the proteasome inhibitors of theinvention can be provided in unit dosage forms.

Thus, the proteasome inhibitors are useful for treating such conditionsas tissue rejection, arthritis, local infections, dermatoses,inflammatory bowel diseases, autoimmune diseases, etc. The proteasomeinhibitors of the present invention can be employed to prevent therejection or inflammation of transplanted tissue or organs of any type,for example, heart, lung, kidney, liver, skin grafts, and tissue grafts.

Compounds of the present invention inhibit the growth of cancer cells.Thus, the compounds can be employed to treat cancer, psoriasis,restenosis or other cell proliferative diseases in a patient in needthereof.

By the term “treatment of cancer” or “treating cancer” is intendeddescription of an activity of compounds of the present invention whereinsaid activity prevents or alleviates or ameliorates any of the specificphenomena known in the art to be associated with the pathology commonlyknown as “cancer.” The term “cancer” refers to the spectrum ofpathological symptoms associated with the initiation or progression, aswell as metastasis, of malignant tumors. By the term “tumor” isintended, for the purpose of the present invention, a new growth oftissue in which the multiplication of cells is uncontrolled andprogressive. The tumor that is particularly relevant to the invention isthe malignant tumor, one in which the primary tumor has the propertiesof invasion or metastasis or which shows a greater degree of anaplasiathan do benign tumors.

Thus, “treatment of cancer” or “treating cancer” refers to an activitythat prevents, alleviates or ameliorates any of the primary phenomena(initiation, progression, metastasis) or secondary symptoms associatedwith the disease. Cancers that are treatable are broadly divided intothe categories of carcinoma, lymphoma and sarcoma, examples ofcarcinomas that can be treated by the composition of the presentinvention include, but are not limited to: adenocarcinoma, acinic celladenocarcinoma, adrenal cortical carcinomas, alveoli cell carcinoma,anaplastic carcinoma, basaloid carcinoma, basal cell carcinoma,bronchiolar carcinoma, bronchogenic carcinoma, renaladinol carcinoma,embryonal carcinoma, anometroid carcinoma, fibrolamolar liver cellcarcinoma, follicular carcinomas, giant cell carcinomas, hepatocellularcarcinoma, intraepidermal carcinoma, intraepithelial carcinoma,leptomanigio carcinoma, medullary carcinoma, melanotic carcinoma,menigual carcinoma, mesometonephric carcinoma, oat cell carcinoma,squamal cell carcinoma, sweat gland carcinoma, transitional cellcarcinoma, and tubular cell carcinoma. Sarcomas that can be treated bythe composition of the present invention include, but are not limitedto: amelioblastic sarcoma, angiolithic sarcoma, botryoid sarcoma,endometrial stroma sarcoma, ewing sarcoma, fascicular sarcoma, giantcell sarcoma, granulositic sarcoma, immunoblastic sarcoma, juxaccordialosteogenic sarcoma, coppices sarcoma, leukocytic sarcoma (leukemia),lymphatic sarcoma (lympho sarcoma), medullary sarcoma, myeloid sarcoma(granulocitic sarcoma), austiogenci sarcoma, periosteal sarcoma,reticulum cell sarcoma (histiocytic lymphoma), round cell sarcoma,spindle cell sarcoma, synovial sarcoma, and telangiectatic audiogenicsarcoma. Lymphomas that can be treated by the composition of the presentinvention include, but are not limited to: Hodgkin's disease andlymphocytic lymphomas, such as Burkitt's lymphoma, NPDL, NML, NH anddiffuse lymphomas.

The compounds of formulae (1b) and (2b) appear to be particularly usefulin treating metastases.

Amounts and regimens for the administration of proteasome inhibitors andcompositions of the invention can be determined readily by those withordinary skill in the clinical art of treating cancer-related disorderssuch as the primary phenomena (initiation, progression, metastasis) orsecondary symptoms associated with the disease. Generally, the dosage ofthe composition of the invention will vary depending upon considerationssuch as: type of composition employed; age; health; medical conditionsbeing treated; kind of concurrent treatment, if any, frequency oftreatment and the nature of the effect desired; extent of tissue damage;gender; duration of the symptoms; and, counter indications, if any, andother variables to be adjusted by the individual physician. A desireddosage can be administered in one or more applications to obtain thedesired results. Pharmaceutical compositions containing the proteasomeinhibitors of the invention can be provided in unit dosage forms.

The present invention will now be illustrated by the following examples,which are not intended to be limiting in any way.

EXAMPLES

Most compounds of formulas (1a), (1b), (2a) or (2b) were preparedaccording to the general reaction sequence depicted in Scheme 1. R² andR³ are as defined above for formulas (1b) and (2b). PG represents anamino-group-protecting moiety. The general procedures employed for eachcompound are summarized in Table I, and detailed descriptions of theseprocedures are provided in the Examples. Syntheses that do not conformto the general reaction sequence are described in full in the Examples.(1S,2S,3R,5S)-Pinanediol leucine boronate trifluoroacetate salt wasprepared as previously reported (Kettner, C. A.; Shenvi, A. B. J. Biol.Chem. 259:15106 (1984)). N-Protected (Boc-, Cbz-, or Fmoc-) amino acidswere commercially available or were prepared from the corresponding freeamino acid by standard protection methods, unless otherwise described inthe Examples. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (EDC), benzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate (BOP reagent), orO-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU) were employed as coupling reagents (Sheehan, J. C. et al., J. Am.Chem. Soc. 87:2492 (1965); Castro, B., et al., Synthesis 11:751 (1976);Tetrahedron Lett. 30:1927 (1989)). All compounds were characterized byproton nuclear magnetic resonance (NMR) spectroscopy. The purity of theproducts was verified by thin layer chromatography and by highperformance liquid chromatography (HPLC).

TABLE I Synthesis of Boronic Ester and Acid Compounds Boronic AcidN-Terminal Compound Coupling Agent Deprotection^(a) Protection MG-261EDC — — MG-262 EDC A — MG-264 BOP — — MG-267 EDC — — MG-268 EDC A NaH,MeI MG-270 EDC A — MG-272 EDC A — MG-273 EDC A, B RC(O)Cl MG-274 BOP A —MG-278 EDC A RC(O)Cl MG-282 EDC A — MG-283 BOP A Ac₂O MG-284 — B RC(O)ClMG-285 BOP A RC(O)Cl MG-286 EDC A, B RC(O)Cl MG-287 EDC B Ac₂O MG-288EDC A RC(O)Cl MG-289 EDC B RS(O)₂Cl MG-290 EDC B Ac₂O MG-291 EDC BRS(O)₂Cl MG-292 BOP B RC(O)Cl MG-293 TBTU B RC(O)Cl MG-294 EDC B —MG-295 BOP B RS(O)₂Cl MG-296 EDC B RS(O)₂Cl MG-297 EDC B RS(O)₂Cl MG-298EDC B RC(O)Cl MG-299 EDC B RC(O)Cl MG-300 EDC B RC(O)Cl MG-301 BOP BAc₂O MG-302 EDC B — MG-303 EDC B HCl, ether MG-304 TBTU B — MG-305 EDC BRC(O)Cl MG-306 TBTU B RC(O)Cl MG-307 TBTU B RC(O)Cl MG-308 TBTU BRC(O)Cl MG-309 TBTU B RC(O)Cl MG-310 BOP B Ac₂O MG-311 BOP B HCl,dioxane MG-312 EDC B RC(O)Cl MG-313 — B RCO₂H, TBTU MG-314 TBTU BRC(O)Cl MG-315 BOP B RC(O)Cl MG-316 BOP B MG-319 TBTU B MG-321 TBTU BRC(O)Cl MG-322 TBTU B RC(O)Cl MG-323 — B Ac₂O MG-325 TBTU B RCO₂H, TBTUMG-328 TBTU B RC(O)Cl MG-329 TBTU B RC(O)Cl MG-332 TBTU B NaH, MeIMG-333 TBTU B NaH, MeI MG-334 TBTU B NaH, MeI MG-336 TBTU B RC(O)ClMG-337 TBTU B HCl, dioxane MG-338 EDC B RC(O)Cl MG-339 TBTU B HCl,dioxane MG-340 TBTU B HCl, dioxane MG-341 TBTU B RCO₂H, TBTU MG-342 — BRNH₂, TBTU MG-343 TBTU B RCO₂H, TBTU MG-344 BOP B Ac₂O MG-345 EDC BRC(O)Cl MG-346 EDC B RC(O)Cl MG-347 EDC B RS(O)₂Cl MG-348 TBTU B HCl,dioxane MG-349 TBTU B HCl, dioxane MG-350 TBTU B PhCH₂NCO MG-351 EDC B —MG-352 TBTU B RCO₂H, TBTU MG-353 TBTU B RC(O)Cl MG-354 BOP B RS(O)₂ClMG-356 TBTU B — MG-357 TBTU B HCl, dioxane MG-358 TBTU B RC(O)Cl MG-359TBTU B HCl, dioxane MG-361 TBTU B RCO₂H, TBTU MG-362 — B PhCH₂NCO MG-363TBTU B HCl, dioxane MG-364 — B RCO₂H, TBTU MG-366 TBTU B HCl, dioxaneMG-367 — B RC(O)Cl MG-368 EDC B TBTU MG-369 TBTU B HCl, dioxane MG-380TBTU B RS(O)₂Cl MG-382 TBTU B RCO₂H, TBTU MG-383 TBTU B RCO₂H, TBTUMG-385 TBTU B HCl, dioxane MG-386 TBTU B HCl, dioxane MG-387 TBTU BRC(O)Cl ^(a)A = NaIO₄, NH₄OAc, acetone-water; B = i-BuB(OH)₂, 1N HCl,MeOH-hexane. See Examples for detailed descriptions of procedures.

Example 1 N-(4-Morpholine)carbonyl-β-(1-naphthyl)-L-alanine-L-leucineboronic acid [MG-273] A. (1S,2S,3R,5S)-PinanediolN-Boc-β-(1-naphthyl)-L-alanine-L-leucine boronate

To a solution of (1S,2S,3R,5S)-pinanediol leucine boronatetrifluoroacetate salt (664 mg, 1.76 mmol) andN-Boc-β-(1-naphthyl)-L-alanine (555 mg, 1.76 mmol) in DMF (10 mL) at 0°C. was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(EDC) (404 mg, 2.11 mmol), 1-hydroxybenzotriazole monohydrate (HOBT)(285 mg, 2.11 mmol), and N-methylmorpholine (NMM) (0.3 mL, 2.64 mmol).The mixture was allowed to warm to room temperature and stir overnight.The reaction was quenched with water (100 mL), and the mixture wasextracted with CH₂Cl₂ (4×25 mL). The combined organic layers were washedwith 5% aqueous HCl and saturated aqueous NaHCO₃, dried over anhydrousMgSO₄, filtered, and concentrated to give a yellow oil. Water was addedand the resultant gummy precipitate was extracted with ether (3×25 mL).The organic layer was dried (anhydrous MgSO₄), filtered, andconcentrated to afford the title compound (202 mg) as a white foam.

B. (1S,2S,3R,5S)-Pinanediol β-(1-Naphthyl)-L-alanine-L-leucine boronatetrifluoroacetate salt

To a solution of the product of Example 1A (930 mg, 1.38 mmol) in CH₂Cl₂(10 mL) at 0° C. was added trifluoroacetic acid (5 mL) and thioanisole(1 mL). The reaction mixture was allowed to warm to room temperature.After 4 h, the reaction mixture was concentrated to dryness and dried invacuo. The residue was used in the next reaction without furtherpurification.

C. (1S,2S,3R,5S)-PinanediolN-(4-morpholine)carbonyl-β-(1-naphthyl)-L-alanine-L-leucine boronate

4-Morpholinecarbonyl chloride (50 mL, 0.42 mmol) and triethylamine (150mL, 1.08 mmol) were added to a solution of the product of Example 1B(0.25 g, 0.36 mmol) in CH₂Cl₂ (6 mL). After 24 h, additionalmorpholinecarbonyl chloride (50 mL) and triethylamine (150 mL) wereadded. After 2 days total reaction time, the reaction mixture wasdiluted with EtOAc, washed with 1N HCl and saturated aqueous NaHCO₃,dried over MgSO₄, filtered, and concentrated. Purification by flashchromatography (elution with 1:2 EtOAc/hexanes and 4:4:1hexanes/EtOAc/MeOH) afforded the title compound (124 mg).

D. N-(4-Morpholine)carbonyl-β-(1-naphthyl)-L-alanine-L-leucine boronicacid

To a stirred solution of the product of Example 1C (124 mg, 0.21 mmol)in acetone (10 mL) was added aqueous NH₄OAc (0.1 N, 5 mL, 1.0 mmol),followed by NaIO₄ (120 mg, 0.21 mmol). The reaction mixture was stirredat room temperature for 72 h, and then the acetone was evaporated. Theaqueous layer was acidified to pH 3 with 1N HCl and extracted with EtOAc(3×20 mL). The combined organic layers were dried over anhydrous MgSO₄,filtered, and concentrated. The residue was purified by flashchromatography (elution with 1:1 hexane/EtOAc, 2:2:1 hexanes/EtOAc/MeOH,and 1:1 few drops MeOH:EtOAc:HOAc) to give the title compound (29 mg).

Example 2 N-Cbz-L-Leucine-L-leucine boronic acid [MG-274] A.(1S,2S,3R,5S)-Pinanediol N-Cbz-L-leucine-L-leucine boronate

Benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(BOP reagent, 827 mg, 1.87 mmol) was added in one portion to a mixtureof (1S,2S,3R,5S)-pinanediol leucine boronate trifluoroacetate salt (595mg, 1.58 mmol), N-Cbz-L-leucine (500 mg, 1.87 mmol) in acetonitrile (30mL) at room temperature. The mixture was stirred at room temperature for2 hours. The reaction was quenched with brine (50 mL) and the mixturewas extracted with EtOAc (3×50 mL). The combined organic layers werewashed with aqueous 5% HCl, saturated aqueous NaHCO₃, and saturatedaqueous NaCl, and then dried (anhydrous MgSO₄), filtered, andconcentrated. The residue was purified by silica gel chromatography(elution with 20–30% acetone/hexanes) to afford the title compound (539mg).

B. N-Cbz-L-Leucine-L-leucine boronic acid

By a procedure analogous to that described in Example 1D, the compoundof Example 2A above (539 mg) was deprotected by treatment with sodiummetaperiodate (1.2 g, 5.61 mmol) and aqueous NH₄OAc (0.1 N, 10 mL, 1.0mmol) to provide the title compound as a white solid (154 mg).

Example 3 β-(1-Naphthyl)-L-alanine-L-leucine boronic acid hydrochloridesalt [MG-302] and β-(1-Naphthyl)-L-alanine-L-leucine boronic acid[MG-303] A. (1S,2S,3R,5S)-Pinanediol β-(1-naphthyl)-L-alanine-L-leucineboronate hydrochloride salt

To a solution of (1S,2S,3R,5S)-pinanediolβ-(1-naphthyl)-L-alanine-L-leucine boronate trifluoroacetate salt(prepared as described in Example 1B, 536 mg, 0.93 mmol) in ether (2 mL)was added 10 mL of 1N HCl. The mixture was sonicated for severalminutes. Ether was allowed to slowly evaporate. The resultant crystalswere collected, washed with H₂O and ether, and dried in vacuo to providethe title compound (300 mg).

B. β-(1-Naphthyl)-L-alanine-L-leucine boronic acid hydrochloride salt;and β-(1-Naphthyl)-L-alanine-L-leucine boronic acid

To the product of Example 3A (290 mg, 0.58 mmol) in a mixture of hexane(4 mL), MeOH (4 mL), and 1N HCl (1.3 mL) was added i-BuB(OH)₂ (71 mg,0.70 mmol). The reaction mixture was stirred for 72 h at roomtemperature. The MeOH-H₂O layer was washed with hexanes, and the MeOHwas evaporated. The aqueous solution was made basic with NaOH and washedwith ether-EtOAc (1:1). The aqueous layer was lyophilized to give 640 mgof a yellow solid. The solid was dissolved in MeOH, 4N HCl in1,4-dioxane was added, and the solution was filtered to remove a whitesolid. The filtrate was concentrated and the residue was purified byreverse phase HPLC (elution with CH₃CN—H₂O) to afford 45 mg of MG-302and 10 mg of MG-303.

Example 4 N-(4-Morpholine)carbonyl-(O-benzyl)-L-tyrosine-L-leucineboronic acid [MG-306] A. N-Boc-O-Benzyl-L-tyrosine

A suspension of O-benzyl-L-tyrosine (3.12 g, 11.5 mmol) in a mixture of1,4-dioxane (14 mL) and water (14 mL) was treated, in order, withtriethylamine (5.0 mL, 35.9 mmol) and a solution of (Boc)₂O (2.86 g,13.1 mmol) in 1,4-dioxane (12 mL). After 19 h, the reaction mixture wasdiluted with water (140 mL) and washed with ether. The aqueous layer wasacidified with 1N citric acid (35 mL) and extracted with CH₂Cl₂ (2×100mL). Additional citric acid (15 mL) was added to the aqueous layer,which was again extracted with CH₂Cl₂ (100 mL). The combined organicextracts were dried (MgSO₄), filtered, and concentrated to give thecrude product (4.5 g), which was used directly in the next reaction.

B. (1S,2S,3R,5S)-Pinanediol N-Boc-(O-benzyl)-L-tyrosine-L-leucineboronate

To a stirred and cold (0° C.) solution of (1S,2S,3R,5S)-pinanediolβ-(1-naphthyl)-L-alanine-L-leucine boronate trifluoroacetate salt(prepared as described in Example 1B, 3.03 g, 7.98 mmol),N-Boc-O-benzyl-L-tyrosine (2.97 g, 7.99 mmol), and TBTU (3.35 g. 8.84mmol) in anhydrous DMF (30 mL) was added by syringe pump, at the rate of1.9 mL/h, DIEA (4.2 mL, 24.1 mmol). After the addition was complete, themixture was allowed to warm to room temperature over 30 min, and then itwas added dropwise to 30 mL of rapidly stirring water. Additional waterwas added and the mixture was filtered. The collected solid wasdissolved in MeOH, concentrated to near dryness and again added torapidly stirring water (300 mL). The resultant white solid was collectedby suction filtration, washed with water, frozen, and lyophilized toprovide the title compound (4.49 g).

C. (1S,2S,3R,5S)-Pinanediol (O-benzyl)L-tyrosine-L-leucine boronate

The product of Example 4B (4.47 g, 7.23 mmol) was dissolved in CH₂Cl₂(40 mL) and cooled to 0° C. A solution of 4N HCl in dioxane (40 mL, 0.16mol) was added and the reaction mixture was stirred at room temperaturefor 1.5 h. Concentration afforded a yellow solid, which was trituratedwith hexane-ether (1:1, 100 mL). Filtration afforded the title compound(3.65 g) as a pale yellow solid.

D. (1S,2S,3R,5S)-PinanediolN-(4-morpholine)carbonyl-(O-benzyl)-L-tyrosine-L-leucine boronate

By a procedure analogous to that described in Example 1C, the product ofExample 4C (2.53 g, 4.56 mmol) was treated with 4-morpholinecarbonylchloride (0.75 mL, 6.43 mmol) to provide the title compound (2.35 g) asa pale yellow solid.

E. N-(4-morpholine)carbonyl-(O-benzyl)-L-tyrosine-L-leucine boronic acid

The product of Example 4D (0.39 g, 0.62 mmol) was deprotected accordingto the procedure described in Example 3B to provide the title compound(146 mg) as a white solid.

Example 5 N-Methyl-N-Cbz-L-leucine-L-leucine boronic acid [MG-268] A.N-Methyl-N-Cbz-L-leucine

To a solution of N-Cbz-leucine (1.38 g, 5.2 mmol) in THF (15 mL) at 0°C. was added methyl iodide (2.5 mL, 40.1 mmol). Sodium hydride (60%dispersion in oil, 0.6 g, 15 mmol) was added cautiously, and theresultant mixture was stirred at room temperature for 24 h. The reactionmixture was diluted with EtOAc (25 mL) and water (2 mL) was addeddropwise. The mixture was concentrated to dryness, and the residue waspartitioned between ether (15 mL) and water (50 mL). The organic layerwas extracted with saturated aqueous NaHCO₃ (25 mL), and the combinedaqueous extracts were acidified to pH 2 with 3N HCl. The product wasextracted with EtOAc (3×25 mL), dried over MgSO₄, filtered, andconcentrated to afford the title compound (1.41 g) as a yellow solid.

B. (1S,2S,3R,5S)-Pinanediol-N-methyl-N-Cbz-L-leucine-L-leucine boronate

By a procedure analogous to that described in Example 1A, the product ofExample 5A (85.1 mg, 0.30 mmol) was coupled with(1S,2S,3R,5S)-pinanediol leucine boronate trifluoroacetate salt (105 mg,0.28 mmol) in the presence of EDC (64 mg, 0.33 mmol), HOBT (45 mg, 0.33mmol), and NMM (37 mg, 0.37 mmol) to provide, after purification byflash chromatography (elution with 3:2 hexanes/acetone), the titlecompound (85 mg).

C. N-Methyl-N-Cbz-L-leucine-L-leucine boronic acid

By a procedure analogous to that described in Example 1D, the product ofExample 5B (85 mg, 0.16 mmol) was deprotected by treatment with NaIO₄(104 mg, 0.485 mmol) and aqueous NH₄OAc (0.1N, 5 mL, 0.5 mmol) in 10 mLof acetone to provide, after purification by flash chromatography(elution with 4:4:2 hexanes/acetone/MeOH), the title compound (21 mg).

Example 6N-(4-Morpholine)carbonyl-β-(6-quinolinyl)-D,L-alanine-L-leucine boronicacid [MG-292] A. β-(6-Quinolinyl)-D,L-alanine

N-Acetyl β-(6-quinolinyl)-D,L-alanine ethyl ester (728 mg, 2.55 mmol)was heated at reflux in 6N HCl (20 mL). After 20 h, the reaction mixturewas concentrated to dryness and the residue was dried in vacuo toprovide the title compound, which was used directly in the nextreaction.

B. N-Boc-β-(6-Quinolinyl)-D,L-alanine

To the crude product of Example 6A in a stirred mixture of 1,4-dioxane(10 mL), water (10 mL), and 2N NaOH (5 mL) at 0° C. was addeddi-tert-butyl pyrocarbonate (556 mg, 2.55 mmol). The reaction mixturewas allowed to warm to room temperature. After 23 h, the reactionmixture was acidified to pH 4 and extracted with EtOAc (3×50 mL) andn-BuOH (3×50 mL). The combined extracts were concentrated to provide thetitle compound, which was used directly in the next reaction.

C. (1S,2S,3R,5S)-Pinanediol N-Boc-β-(6-quinolinyl)-D,L-alanine-L-leucineboronate

By a procedure analogous to that described in Example 2A, the product ofExample 6B was coupled with (1S,2S,3R,5S)-pinanediol leucine boronatetrifluoroacetate salt (943 mg, 2.5 mmol) in the presence of BOP reagent(1.33 g, 3 mmol) and triethylamine (0.37 mL, 2.62 mmol) to provide thetitle compound (343 mg).

D. (1S,2S,3R,5S)-Pinanediol β-(6-quinolinyl)-D,L-alanine-L-leucineboronate

The product of Example 6C (343 mg, 0.61 mmol) was treated withtrifluoroacetic acid (7 mL) and thioanisole (1 mL) in CH₂Cl₂ (15 mL) at0° C., as described in Example 1B, to provide the title compound.

E. (1S,2S,3R,5S)-PinanediolN-(4-morpholine)carbonyl-β-(6-quinolinyl)-D,L-alanine-L-leucine boronate

The product of Example 6D was coupled with 4-morpholinecarbonyl chloride(0.14 mL, 1.22 mmol) by a procedure analogous to that described inExample 1C to produce the title compound (112 mg).

F. N-(4-Morpholine)carbonyl-β-(6-quinolinyl)-D,L-alanine-L-leucineboronate

Deprotection of the product of Example 6E (153 mg, 0.27 mmol) waseffected according to the procedure described in Example 3B.Purification by silica gel chromatography (elution with 50:50:10hexanes/acetone/methanol) afforded the title compound (87 mg). Theproduct was further purified by reverse phase HPLC; 5 mg of the titlecompound was recovered.

Example 7 N-(4-Morpholine)carbonyl-β-(1-naphthyl)-L-alanine-L-leucinemethylboronic acid [MG-317]; andN-(4-Morpholine)carbonyl-β-(1-naphthyl)-L-alanine-L-leucinedimethylborane [MG-318]

To a suspension of MG-273 (prepared as described in Example 1, 101.5 mg,0.23 mmol) in 3 mL of a 2:1 mixture of Et₂O/CH₂Cl₂ was added1,3-propanediol (20.0 mL, 0.28 mmol). The resultant clear solution wasstirred for 30 min at room temperature, and then anhydrous MgSO₄ wasadded. Stirring was continued for an additional 30 min, and then themixture was filtered through a cotton plug and then through a 0.2 mmPTFE filter. The solution was concentrated, toluene (2 mL) was added,and the mixture was again concentrated to produce a white solid.Anhydrous THF (3 mL) was added, and the resultant solution was cooled to0° C. MeLi (0.8 mL, 1.12 mmol) was added. After 10 min, the mixture waswarmed to room temperature. After 20 min, the light red solution wascooled to 0° C., quenched with a few drops of water, and then dilutedwith 10 mL of 1N HCl. The colorless solution was extracted with CH₂Cl₂(2×10 mL), and the combined extract was concentrated to afford a whitesolid. Purification by flash chromatography (elution with 2–4%MeOH/HCl₃, followed by 10% MeOH/CH₃) afforded MG-317 (17.7 mg) andMG-318 (72.1 mg).

Example 8 N-Benzyl-(3R)-3-dioxyboryl-5-methylhexanamide [MG-342] A.tert-Butyl-(3R)-3-[(1S,2S,3R,5S)-(pinanediyldioxy)boryl]-5-methylhexanoate

A 200-mL round-bottomed flask was charged with anhydrous THF (50 mL) andtert-butyl acetate (0.48 mL, 3.56 mmol). The solution was cooled to −78°C. under nitrogen, and LDA (1.5 M solution in cyclohexane, 2.2 mL, 3.3mmol) was added by syringe over 8 min. The resultant solution wasstirred for 10 min, and then a solution of (1S,2S,3R,5S)-pinanediol1-bromo-3-methylbutylboronate (Organometallics 9:3171 (1990)) (1.04 g,3.15 mmol) in ahhydrous THF (15 mL) was added by cannula over 8 min. Thereaction mixture was allowed to warm to room temperature and stirovernight. The pale pink solution was concentrated, and the residue wasdissolved in 200 mL of ether. The solution was washed with saturatedaqueous NH₄Cl and saturated aqueous NaCl. Concentration gave a clearorange oil, which was purified by flash chromatography (elution with2–3% EtOAc/hexanes) to afford the title compound (584 mg).

B. (3R)-3-[(1S,2S,3R,5S)-(pinanediyldioxy)boryl]-5-methylhexanoic acid

To a solution of the product of Example 8A (323 mg, 0.89 mmol) in CH₂Cl₂(8 mL) was added trifluoroacetic acid (2.0 mL, 26 mmol). The resultantmixture was stirred at room temperature for 2 h. The reaction mixturewas concentrated and dried overnight under high vacuum to produce a darkbrown oil (309.3 mg).

C.N-Benzyl-(3R)-3-[(1S,2S,3R,5S)-pinanediyldioxy)boryl]-5-methylhexanamide

To a solution of the product of Example 8B (300 mg, 0.9 mmol) and TBTU(410 mg, 1.08 mmol) in anhydrous acetonitrile (5 mL) was addedbenzylamine (0.12 mL, 1.10 mmol), followed by diisopropylethylamine(0.50 mL, 2.9 mmol). The reaction mixture was stirred overnight at roomtemperature, and then was poured into water and extracted with EtOAc.The organic layer was washed with saturated aqueous NaHCO₃ and saturatedaqueous NaCl. Concentration gave a dark brown oil, which was purified byflash chromatography (elution with 20% EtOAc/hexanes) to afford thetitle compound (232 mg) as a clear, colorless oil.

D. N-Benzyl-(3R)-3-dioxyboryl-5-methylhexanamide

The product of Example 8C (223 mg, 0.56 mmol) was deprotected accordingto the procedure described in Example 3B. Purification by flashchromatography (elution with 5% MeOH/CHCl₃) provided a pale yellow oil,which was dissolved in acetonitrile/MeOH. Water was added and themixture was lyophilized overnight to produce the title compound (108 mg)as a fluffy white solid.

Example 9 N-Acetyl-1,2,3,4-tetrahydro-3-isoquinolinecarbonyl-L-leucineboronic acid [MG-310] A.N-Boc-1,2,3,4-Tetrahydro-3-isoquinolinecarboxylic acid

A solution of 1,2,3,4-tetrahydro-3-isoquinolinecarboxylic acid (855 mg,4.83 mmol), (Boc)₂O (1.37 g, 6.28 mmol), and 1N NaOH (6 mL) in a mixtureof t-BuOH (12 mL) and water (12 mL) was stirred overnight at roomtemperature. The reaction mixture was diluted with water (30 mL) andwashed with ether-hexanes (1:1, 2×25 mL). The organic layer wasback-extracted with 10% NaHCO₃. The combined aqueous layers werecarefully acidified to pH 2–3 and extracted with EtOAc (3×30 mL). Thecombined organic extracts were washed with water and saturated aqueousNaCl, dried (MgSO₄), and concentrated to provide the title compound(1.27 g) as a white solid.

B. (1S,2S,3R,5)-PinanediolN-Boc-1,2,3,4-tetrahydro-3-isoquinolinecarbonyl-L-leucine boronate

To a mixture of (1S,2S,3R,5S)-pinanediol-L-leucine boronatetrifluoroacetate salt (1.14 g, 3.03 mmol),N-Boc-1,2,3,4-tetrahydro-3-isoquinolinecarboxylic acid (762 mg, 2.75mmol), and BOP reagent (1.34 g, 3.03 mmol) in DMF (20 mL) was added,over a period of 2 h, DIEA (1.44 mL, 8:25 mmol). The resultant solutionwas stirred for 1 h after addition was complete. The reaction mixturewas poured into water (300 mL) and extracted with EtOAc (3×75 mL). Thecombined organic extracts were washed with dilute aqueous HCl,half-saturated aqueous NaHCO₃, water, and saturated aqueous NaCl, dried(MgSO₄), and concentrated. The residue was purified by flashchromatography (elution with 20% EtOAc-hexanes) to provide the titlecompound (1.04 g) as a white foamy solid.

C. (1S,2S,3R,5S)-Pinanediol1,2,3,4-tetrahydro-3-isoquinolinecarbonyl-L-leucine boronatehydrochloride salt

The product of Example 9B (755 mg) was dissolved in CH₂Cl₂ (10 mL) andcooled to 0° C. A solution of 4N HCl in dioxane (8 mL, 0.03 mol) wasadded and the reaction mixture was stirred at room temperature.Concentration and trituration with ether-hexanes afforded the titlecompound (565 mg) as an off-white solid.

D. (1S,2S,3R,5S)-PinanediolN-acetyl-1,2,3,4-tetrahydro-3-isoquinolinecarbonyl-L-leucine boronate

The product of Example 9C (262 mg, 0.59 mmol) was treated at roomtemperature with Ac₂O (0.085 mL, 0.89 mmol) and DIEA (0.18 mL, 1.36mmol) in CH₂Cl₂ (5 mL). After 24 h, the reaction mixture was dilutedwith CH₂Cl₂ (20 mL), washed with 1N HCl, half-saturated NaHCO₃, andwater, dried (Na₂SO₄), and concentrated. Purification by flashchromatography (elution with EtOAc-hexanes) afforded the title compound(271 mg) as a white foamy solid.

E. N-Acetyl-1,2,3,4-tetrahydro-3-isoquinolinecarbonyl-L-leucine boronicacid

By a procedure analogous to that described in Example 3B, the product ofExample 9D (226 mg, 0.49 mmol) was deprotected to provide the titlecompound (131 mg) as a foamy, oily solid.

Example 10 N-(4-Morpholine)carbonyl-β-(2-quinolyl)-L-alanine-L-leucineboronic acid [MG-315] A. Diethyl (2-quinolylmethyl)acetamidomalonate

To a solution of 2(chloromethyl)quinoline monohydrochloride (5.0 g, 23.4mmol) and diethyl acetamidomalonate (10.1 g, 46.7 mmol) in EtOH (60 mL)was added sodium methoxide (3.78 g, 70 mmol). The reaction mixture washeated at reflux for 6 h. The reaction mixture was cooled, filtered, andconcentrated. The residue was dissolved in EtOAc (400 mL) and extractedwith cold 4N HCl (3×150 mL). The aqueous layer was neutralized with 10NNaOH and extracted with EtOAc (3×200 mL). The combined organic extractwas washed with water, dried (anhydrous MgSO₄), filtered, andconcentrated to give the title compound (8.3 g).

B. N-Acetyl-β-(2-quinolyl)-D,L-alanine ethyl ester

To a solution of the product of Example 10A (8 g, 22.3 mmol) in EtOH(180 mL) was added 6.1 N NaOH (6.5 mL, 40 mmol). After 2 h, 11.1N HCl(3.6 mL, 40 mmol) was added, and the reaction mixture was concentratedto dryness. The residue was suspended in 1,4-dioxane (200 mL) and themixture was heated at reflux for 90 min. The reaction mixture wasconcentrated and the residue was purified by silica gel chromatography(elution with 30–50% acetone-hexanes) to provide to title compound (4.3g).

C. N-Acetyl-β-(2-quinolyl)-L-alanine

The product of Example 10B (4.3 g, 15 mmol) was treated with SubtilisinCarlsberg (Sigma, 11.9 units/mg, 30 mg, 357 units) at room temperaturein aqueous NaHCO₃ (0.2M, 120 mL). After 2 h, the reaction mixture wasextracted with CHCl₃ (6×100 mL). The aqueous layer was concentrated todryness to provide the title compound (3.5 g), which contained salts.

D. N-Boc-β-(2-Quinolyl)-L-alanine

A solution of the product of Example 10C (3.5 g, ca. 7.4 mmol) in 6N HCl(40 mL) was heated at reflux for 16 h. The solvent was removed and theresidue was dried in vacuo.

To this residue was added 1,4-dioxane (20 mL), water (20 mL), and 2NNaOH (10 mL, 20 mmol). The solution was cooled to 0° C. and di-t-butylpyrocarbonate (1.6 g, 7.5 mmol) was added. After 1 h at 0° C., thereaction mixture was warmed to room temperature and stirring wascontinued for 17 h. The reaction mixture was extracted with CH₂Cl₂ (100mL) and n-BuOH (4×100 mL). The aqueous layer was acidified and againextracted with n-BuOH. The organic extracts were combined andconcentrated to provide the title compound (1.6 g).

E. (1S,2S,3R,5S)-Pinanediol N-Boc-β-(2-quinolyl)-L-alanine-L-leucineboronate

By a procedure analogous to that described in Example 2A, the product ofExample 10D (0.6 g, 1.9 mmol) was coupled with (1S,2S,3R,5S)-pinanediolleucine boronate trifluoroacetate salt (716 mg, 1.9 mmol) in thepresence of BOP reagent (0.84 g, 1.9 mmol) and triethylamine (0.27 mL,1.9 mmol). Purification by silica gel chromatography (elution with10–30% acetone-hexanes) afforded the title compound (194 mg).

F. (1S,2S,3R,5S)-PinanediolN-(4-morpholine)carbonyl-β-(2-quinolyl-L-alanine-L-leucine boronate

The product of Example 10E (194 mg) was treated with trifluoroaceticacid (7 mL) and thioanisole (1 mL) as described in Example 1B. Theresultant product was condensed with 4-morpholinecarbonyl chloride (568mg, 3.8 mmol) as described in Example 2C. Purification by silica gelchromatography (elution with 20–50% acetone-hexanes) afforded the titlecompound (367 mg).

G. N-(4-Morpholine)carbonyl-β-(2-quinolyl)-L-alanine-L-leucine boronicacid

The product of Example 10F (367 mg, 0.64 mmol) was deprotected accordingto the procedure described in Example 3B to provide the title compound(222 mg).

Example 11 N-Boc-1,2,3,4-tetrahydro-1-isoquinolinecarboxylic acid[precursor for the synthesis of MG-310] A.1,2,3,4-Tetrahydro-1-isoquinolinecarboxylic acid

A solution of 1-isoquinolinecarboxylic acid (1.67 g) in glacial aceticacid (25 mL) was hydrogenated at 60 p.s.i. over PtO₂ (270 mg). When thereaction was complete, the mixture was filtered through diatomaceousearth (Celite), washing the solid pad with MeOH, and the filtrate wasconcentrated to dryness. The resultant white solid was triturated withcold water and filtered to provide the title compound (775 mg).

B. A-Boc-1,2,3,4-tetrahydro-1-isoquinolinecarboxylic acid

The product of Example 11B (762 mg, 4.3 mmol) was treated withdi-tert-butyl pyrocarbonate (1.13 g, 5.17 mmol) according to theprocedure described in Example 6B to afford the title compound (886 mg),as a foamy white solid.

Example 12 DiethanolamineN-(4-morpholine)carbonyl-β-(1-naphthyl)-L-alanine-L-leucine boronate[MG-286]

To a solution ofN-(4-morpholine)carbonyl-β-(1-naphthyl)-L-alanine-L-leucine boronic acid(prepared as described in Example 1, 97.4 mg, 0.22 mmol) in CH₂Cl₂ (4mL) was added a solution of diethanolamine (25.5 mg, 0.24 mmol) in EtOAc(1 mL). The resultant solution was stirred at room temperature for 0.5h. Anhydrous Na₂SO₄ (1.5 g) was added and stirring was continued for anadditional 0.5 h. The reaction mixture was filtered and concentrated,and the crude product was purified by stirring in hot EtOAc (2 mL) andprecipitation with hexanes (1 mL). The solid was collected, washed withhexanes, and dried to provide the title compound (106 mg).

Example 13N-[3-(4-morpholine)carbonyl-2(R)-(1-naphthyl)methyl]propionyl-L-leucineboronic acid [MG-324] A. 1-Naphthalenecarboxaldehyde

To a cold (−78° C.) solution of oxalyl chloride (6.9 mL, 0.079 mol) indry CH₂Cl₂ (200 mL) was added dropwise dry DMSO (11.2 mL, 0.158 mol).The mixture was stirred for 10 min, and then a solution of1-naphthalenemethanol (10.0 g, 0.063 mol) in dry CH₂Cl₂ (40 mL) wasadded over 15 min. The mixture was stirred for 10 min, and then Et₃N (44mL, 0.316 mol) was added slowly. The reaction mixture was allowed towarm to room temperature. After 3.5 h, to the pale yellow heterogeneousmixture was added 10% aqueous citric acid (30 mL) and water (100 mL).The organic phase was washed with water (100 mL) and saturated aqueousNaCl (100 mL), dried (anhydrous MgSO₄), filtered, and concentrated.Ether-hexane (1:1) was added, and the mixture was filtered.Concentration provided a pale orange oil (9.7 g).

B. Ethyl 3-(1-naphthyl)propenoate

To a solution of the product of Example 12A (9.7 g, 62 mmol) in CH₂Cl₂(150 mL) was added at room temperature (carbethoxymethylene)triphenylphosphorane (25 g, 71 mmol). The resultant mixture was stirredfor 1.5 h, and the homogeneous yellow solution was then concentrated todryness. Ether-hexane (1:1) was added, the mixture was filtered, and thefiltrate was concentrated to dryness to provide a pale orange oil (15.3g).

C. Ethyl 3-(1-naphthyl)propionate

The product of Example 12B (15.3 g, 68 mmol) was dissolved in a mixtureof EtOAc (100 mL) and MeOH (10 mL) and hydrogenated at 1 atm. over 10%Pd/C (0.5 g). The reaction was continued for 4 days, replacing thecatalyst with fresh catalyst several times. The reaction mixture wasfiltered and concentrated to provide 13 g of a crude oil.

D. 3-(1-Naphthyl)propionic acid

To a solution of the product of Example 12C (13 g) in a mixture of THF(100 mL) and water (25 mL) was added 1N NaOH (75 mL, 75 mmol). The brownreaction mixture was stirred at room temperature overnight. The THF wasremoved, and the aqueous layer was washed with ether (2×50 mL). Theaqueous layer was acidified to pH 2 with 6N HCl and the precipitatedsolid was collected, washed with water (100 mL), and lyophilized to give9.3 g of a pale yellow solid.

E. 3-(1-Naphthyl)propionyl chloride

To a suspension of the product of Example 12D (4.0 g, 20 mmol) in CH₂Cl₂(25 mL) at 0° C. was added oxalyl chloride (1.9 mL, 22 mmol) and DMF(0.1 mL). The reaction mixture was warmed to room temperature and thenheated with a heat gun. Additional oxalyl chloride (0.5 mL) was addedand heating was continued to produce a dark homogeneous mixture. Thereaction mixture was concentrated, the residue was redissolved inCH₂Cl₂-hexane, and the resultant solution was filtered. Concentrationafforded 4.9 g of a green liquid.

F. 4(S)-Isopropyl-3-[3-(1-naphthyl)-1-oxopropyl]-2-oxazolidinone

To a solution of (4S)(−)-4-isopropyl-2-oxazolidinone (2.32 g, 18 mmol)in dry THF (50 mL) at −78° C. was added dropwise n-BuLi (2.5M inhexanes, 8 mL, 20 mmol). The heterogeneous white mixture was stirred at−78° C. for 30 min, and then a solution of the product of Example 12E(4.9 g, 20 mmol) in dry THF (25 mL) was added dropwise over 15–20 min.After 1.5 h, the reaction was quenched by the addition of 1N HCl (25 mL)and saturated aqueous NaCl (25 mL). The mixture was stirred at roomtemperature for 30 min, and then the THF was removed by rotaryevaporation. The aqueous layer was extracted with EtOAc, and thecombined organic extract was dried (anhydrous MgSO₄), filtered, andconcentrated. The residue was filtered through a pad of silica gel(elution with 20% EtOAc-hexanes) to provide 2.8 g of a pale pink solid.

G.3-[3-Benzyloxycarbonyl-2(R)-[1-naphthyl)methyl]-1-oxopropyl]-4(S)-isopropyl-2-oxazolidinone

To a solution of 1,1,1,3,3,3-hexamethyldisilazane (0.75 mL, 3.5 mmol) indry THF (10 mL) at 0° C. was added n-BuLi (2.5M in hexanes, 1.45 mL, 3.6mmol). After 10 min, the mixture was cooled to −78° C. and a solution ofthe product of Example 12F (1.0 g, 3.2 mmol) in dry THF (8 mL) was addeddropwise. After 30–40 min, benzyl bromoacetate (0.75 mL, 4.8 mmol) wasadded. The mixture was stirred at −78° C. for 1 h, and at 0° C. for 5–10min. The reaction was quenched by the addition of 1N HCl (10 mL), andthe solution was extracted with ether. The combined organic extract waswashed with saturated aqueous NaHCO₃ and saturated aqueous NaCl, driedanhydrous MgSO₄), filtered and concentrated. The wet solid wastriturated with hexane-ether (1:1), filtered, and dried to give thetitle compound (0.6 g) as a white solid.

H.3-[2(R)-(1-naphthyl)methyl]-3-[4(S)-isopropyl-2-oxazolidinoyl]propanoicacid

To the product of Example 12G (600 mg, 1.3 mmol) was added MeOH (15 mL),EtOH (15 mL), EtOAc (5 mL), and CH₂Cl₂ (5 mL), followed by 10% Pd/C (100mg). The reaction mixture was hydrogenated under 1 atm. H₂. The reactionmixture was filtered and concentrated. The residue was triturated withether-hexanes, the solvents were removed, and the resultant white solidwas dried in vacuo to give 480 mg of the title compound.

I.4(S)-Isopropyl-3-[4-morpholino-2(R)-(1-naphthyl)methyl-1,4-dioxobutyl]-2-oxazolidinone

To a solution of the product of Example 12H (473 mg, 1.28 mmol) in dryTHF (25 mL) at 0° C. was added dropwise under nitrogen morpholine (130mL, 1.47 mmol), diethyl pyrocarbonate (240 mL, 1.47 mmol), andtriethylamine (220 mL, 1.6 mmol). After 2 h, the solvent was removed invacuo, and the residue was washed with water and extracted withether-EtOAc (1:1). The combined organic extract was dried (anhydrousMgSO₄), filtered, and concentrated. The residue was triturated withEtOAc-hexanes to provide the title compound (410 mg).

J. 3-(4-morpholine)carbonyl-2(R)-(1-naphthyl)methyl propionic acid

To a solution of the product of Example 12I (400 mg, 0.913 mmol) in amixture of THF (8 mL) and water (2 mL) at 0° C. was added LiOH (80 mg,1.9 mmol). The reaction mixture was stored at 0° C. overnight. Thereaction mixture was concentrated to remove THF, 1N NaOH (20 mL) wasadded, and the mixture was washed with CH₂Cl₂ (15 mL). The aqueous layerwas acidified to pH 2 with 1N HCl and extracted with CH₂Cl₂. Thecombined organic extract was dried (anhydrous MgSO₄), filtered, andconcentrated. The residue was triturated with ether-hexanes, and thesolvents were removed in vacuo to provide the crude product (240 mg) asa white foam.

K.(1S,2S,3R,5S)-PinanediolN-[3-(4-morpholine)carbonyl-2(R)-(1-naphthyl)methyl]propionyl-L-leucineboronate

To a solution of the product of Example 12J (230 mg, 0.7 mmol) in DMF (8mL) at 0° C. was added (1S,2S,3R,5S)-pinanediol leucine boronatetrifluoroacetate salt (293 mg, 0.77 mmol) and TBTU (293 mg, (0.77 mmol).To the resultant mixture was added slowly over 1.5 hdiisopropylethylamine (365 mL, 2.1 mmol). After addition was complete,the reaction mixture was stirred for 30 min. Water (100 mL) was added,and the precipitated solid was collected, washed with water (50 mL), andlyophilized to provide the title compound (300 mg).

L.N-[3-(4-morpholine)carbonyl-2(R)-(1-naphthyl)methyl]propionyl-L-leucineboronic acid

By a procedure analogous to that described in Example 3B, the product ofExample 12K (300 mg, 0.522 mmol) was deprotected to provide the titlecompound (150 mg).

Example 14 trans-4-Phenoxy-L-proline-L-leucine boronic acid [MG-349] A.N-Carbobenzyloxy-(trans-4-hydroxy-L-proline

According to the literature procedure (J. Am. Chem. Soc. 189 (1957)),trans-4-hydroxy-L-proline (5.12 g, 0.039 mol) was treated with benzylchloroformate (8.5 mL, 0.06 mol) to provide the title compound (6.0 g)as a white solid.

B. N-Carbobenzyloxy-trans-4-hydroxy-L-proline methyl ester

To a solution of the product of Example 13A (1.08 g, 3.75 mmol) inacetonitrile (4 mL) at 0° C. was added dropwise DBU (0.62 mL, 4.12mmol). After 5 min, MeI (0.28 mL, 4.5 mmol) was added. The reactionmixture was allowed to warm to room temperature and stir overnight. Thesolvent was removed, the residue was dissolved in ether-EtOAc (1:1, 30mL), and the resultant solution was washed with 1N HCl, dilute aqueousNaHCO₃, water, and saturated aqueous NaCl. The organic layer was dried(anhydrous MgSO₄) and concentrated to provide the title compound (822mg) as a light yellow oil.

C. N-Carbobenzyloxy-trans-4-phenoxy-L-proline methyl ester

To a mixture of the product of Example 13B (495 mg, 1.71 mmol), phenol(193 mg, 2.05 mmol), and triphenylphosphine (537 mg, 2.05 mmol) in THF(7 mL) at 0° C. was added over 1 h diethyl azodicarboxylate (0.32 mL,2.05 mmol) The reaction mixture was allowed to warm to room temperatureand stir overnight. The reaction mixture was concentrated, and theresidue was dissolved in ether (8 mL) and allowed to stand at 0° C.overnight. The solution was decanted and the solids were washed withcold ether. The ethereal solution was concentrated, and the residue waspurified by flash chromatography (elution with 10–30% EtOAc-hexanes) toprovide the title compound (295 mg).

D. N-Carbobenzyloxy-trans-4-phenoxy-L-proline

The product of Example 13C (285 mg, 0.79 mmol) was dissolved in amixture of 0.5N aqueous LiOH (20 mL) and MeOH (10 mL), and the resultantsolution was stirred at room temperature overnight. The MeOH was removedin vacuo, and the aqueous layer was washed with ether (2×20 mL). Theaqueous layer was cooled, acidified with 3N HCl, and extracted withEtOAc (3×20 mL). The combined organic extract was washed with water andsaturated aqueous NaCl, dried (anhydrous MgSO₄), filtered, andconcentrated to provide the title compound (251 mg) as a light yellowsolid.

E: (1S,2S,3R,5S)-pinanediolN-Carbobenzyloxy-trans-4-phenoxy-L-proline-L-leucine boronate

By a procedure analogous to that described in Example 12K, the productof Example 13D (250 mg, 0.72 mmol) was coupled with(1S,2S,3R,5S)-pinanediol leucine boronate trifluoroacetate salt (300 mg,0.79 mmol) in the presence of TBTU (302 mg, 0.79 mmol) to provide thetitle compound (355 mg) as a white solid.

F. (1S,2S,3R,5S)-pinanediol trans-4-phenoxy-L-proline-L-leucine boronate

The product of Example 13E (343 mg) was hydrogenated for 20 h at 1 atm.over 10% Pd/C (45 mg) in EtOH (3 mL). The reaction mixture was filteredthrough Celite and concentrated to provide the title compound (272 mg).

G. trans-4-Phenoxy-L-proline-L-leucine boronic acid

By a procedure analogous to that described in Example 3B, the product ofExample 13F (270 mg, 0.6 mmol) was deprotected to provide the titlecompound (130 mg) as a white solid.

Example 15[(3S,5R)-4-[(8-quinolinesulfonyl)amino]-3-hydroxy-5-(1-naphthyl)pentanoyl]-L-leucineboronic acid A. (4S,5S)-1-Boc-4-hydroxy-5-(1-naphthyl)-pyrrolidin-2-one

To a solution of N-Boc-β-(1-naphthyl)-L-alanine (1.4 g, 4.44 mmol),2,2-dimethyl-1,3-dioxane-4,6-dione (704 mg, 4.88 mmol), and 4-DMAP (1.25g, 10.21 mmol) in CH₂Cl₂ (40 mL) at 0° C. was added isopropenylchloroformate (0.53 mL, 4.8 mmol). The reaction mixture was stirred for1 h at 0° C. and for 2 h at room temperature. The reaction was quenchedby the addition of aqueous KHSO₄. The organic layer was washed withwater, dried (anhydrous MgSO₄), filtered, and concentrated. The residuewas suspended in EtOAc (30 mL) and heated at reflux for 2 h. The solventwas removed in vacuo.

The residue was dissolved in CH₂Cl₂—HOAc (10:1, 30 mL), and sodiumborohydride (310 mg, 8.21 mmol) was added at 0° C. The mixture wasstirred for 1 h at 0° C. and for 15 h at room temperature. Water wasadded, and the organic layer was washed with saturated aqueous NaCl,dried (anhydrous MgSO₄), filtered, and concentrated. Purification bysilica gel chromatography (elution with 20–30% acetone-hexanes) affordedthe title compound (1.24 g).

B.(3S,5R)-4-(tert-butyloxycarbonyl)amino-3-hydroxy-5-(1-naphthyl)pentanoicacid

The product of Example 14B (1.24 g, 3.64 mmol) was dissolved in acetone(15 mL) and aqueous NaOH (1M, 4 mL, 4 mmol) was added. The reactionmixture was stirred at room temperature for 2 h. The mixture wasacidified with 10% HCl and extracted with EtOAc (3×60 mL). The combinedorganic extract was washed with water, dried (anhydrous MgSO₄),filtered, and concentrated. The residue was purified by silica gelchromatography (elution with 30–50% acetone-hexanes and 70:30:10hexane:acetone:methanol) to give the title compound (0.61 g).

C. (1S,2S,3R,5S)-Pinanediol[(3S,5R)-4-(tert-butyloxycarbonyl)amino-3-hydroxy-5-(1-naphthyl)pentanoyl]-L-leucineboronate

By a procedure analogous to that described in Example 2, the product ofExample 14B (395 mg, 1.1 mmol) was coupled with (1S,2S,3R,5S)-pinanediolleucine boronate trifluoroacetate salt (415 mg, 1.1 mol) in the presenceof BOP reagent (487 mg, 1.1 mmol) to afford the title compound (261 mg).

D. (1S,2S,3R,5S)-Pinanediol[(3S,5R)-4-(8-quinolinesulfonyl)amino-3-hydroxy-5-(1-naphthyl)pentanoyl]-L-leucineboronate

The product of Example 14C (261 mg, 0.43 mmol) was dissolved in CH₂Cl₂(10 mL) and treated at 0° C. with trifluoroacetic acid (5 mL) andthioanisole (1 mL). After 2 h, solvents were evaporated.

The residue was dissolved in CH₂Cl₂ (10 mL) and cooled to 0° C.8-Quinolinesulfonyl chloride (98 mg, 0.43 mmol) and triethylamine (0.12mL, 0.86 mmol) were added. The reaction mixture was stirred at 0° C. for1 h and at room temperature for 15 h. The solvents were removed, waterwas added, and the product was extracted with EtOAc (3×50 mL). Thecombined organic extract was washed with saturated aqueous NaHCO₃ andsaturated aqueous NaCl, dried (anhydrous MgSO₄), and concentrated. Theresidue was purified by silica gel chromatography (elution with 20–50%EtOAc-hexanes) to provide the title compound (152 mg).

E.[(3S,5R)-4-(8-quinolinesulfonyl)amino-3-hydroxy-5-(1-naphthyl)pentanoyl]-L-leucineboronic acid

The product of Example 14D (152 mg, 0.22 mmol) was deprotected accordingto the procedure described in Example 3B to provide the title compound(12.7 mg).

Example 16 cis-3-Phenyl-D,L-proline-L-leucine boronic acid hydrochloridesalt [MG-359] A. Diethtyl1-acetyl-4-phenyl-2-pyrrolidinol-5,5-dicarboxylate

Sodium spheres (washed 3× with hexanes and dried in vacuo; 0.13 g, 5.7mmol) were added to a solution of diethyl acetimidomalonate (12.2 g,56.1 mmol) in absolute EtOH under nitrogen. After the sodium haddissolved, the solution was cooled in an ice bath and cinnamaldehyde(7.8 mL, 61.7 mmol) was added dropwise. The bath was removed and thereaction mixture was stirred overnight at room temperature. The solutionwas adjusted to pH 4 with acetic acid (˜3 mL). Solvents were evaporatedand the residue was purified by silica gel chromatography (elution withEtOAc) to give a yellow solid, which was recrystallized (benzene-hexane)to provide the title compound (14.1 g) as a white solid.

B. Diethyl 1-acetyl-3-phenylpyrrolidine-2,2-dicarboxylate

Trifluoroacetic acid (15.4 mL) was added slowly over 15 min to asolution of the product of Example 15A (7.0 g, 20.1 mmol) andtriethylsilane (4.9 mL, 30.8 mmol) in CHCl₃ (40 mL). After 3 h, thesolvents were evaporated and the residue was dissolved in EtOAc (150mL), washed with water, 5% aqueous NaHCO₃, and saturated aqueous NaCl,dried (anhydrous MgSO₄), and concentrated to give 5.9 g of a colorlessoil.

C. N-Acetyl-3-phenylproline ethyl ester

The product of Example 15B (5.9 g) was dissolved in 0.5N NaOH (200 mL)and the resultant solution was stirred at room temperature for 21 h. Thesolution was washed with EtOAc (75 mL) and then acidified to pH 2 with3N HCl. The precipitated solids were extracted with CHCl₃. The organiclayer was concentrated to give a gummy residue, which was dissolved intoluene (70 mL) and heated at 75° C. for 1 h. The solvent was evaporatedto provide the title compound (4.2 g) as a light yellow oil.

D. N-Acetyl-trans-3-phenyl-D,L-proline; andN-acetyl-cis-3-phenyl-D,L-proline ethyl ester

The product of Example 15C (4.2 g, 16 mmol) was dissolved 1M NaOEt inEtOH (100 mL) which contained 2 mL of ethyl trifluoroacetate as a waterscavenger, and the resultant solution was heated at reflux for 2 h. Thereaction mixture was cooled to room temperature, water (65 mL) wasadded, and the solution was stirred for 2.5 h. Most of the EtOH wasremoved by rotary evaporation and the aqueous solution was extractedwith CH₂Cl₂. The aqueous layer was acidified with 3N HCl and extractedwith EtOAc. The organic extract was washed with water and saturatedaqueous NaCl, dried (anhydrous MgSO₄), and concentrated. The orangegummy solid was triturated with ether to provide a yellow solid, whichwas recrystallized (EtOAc-MeOH) to provide the acid (1.91 g) as lightyellow crystals. Concentration of the CH₂Cl₂ extracts afforded the ester(396 mg) as an orange oil.

E. cis-3-Phenyl-D,L-proline hydrochloride salt

The ester obtained in Example 15D (375 mg) was hydrolyzed by heating atreflux in 6N HCl (5 mL) for 17 h. The cooled reaction mixture was washedwith EtOAc and the aqueous layer was concentrated to dryness.Recrystallization (MeOH-ether) afforded the title compound (201 mg).

F. N-Boc-cis-3-Phenyl-D,L-proline

The product of Example 15E (189 mg, 0.84 mmol) was dissolved in amixture of 2N NaOH (3 mL) and 1,4-dioxane (3 mL). tert-Butylpyrocarbonate (218 mg, 1.0 mmol) was added and the reaction mixture wasstirred overnight at room temperature. Dioxane was removed by rotaryevaporation, water (30 mL) was added, and the mixture was washed withEtOAc. The aqueous phase was cooled to 0° C., acidified with 3N HCl, andextracted with EtOAc. The organic layer was washed with water andsaturated aqueous NaCl, dried (anhydrous MgSO₄), and concentrated togive the title compound (199 mg).

G. (1S,2S,3R,5S)-Pinanediol N-Boc-cis-3-phenyl-D,L-proline-L-leucineboronate

By a procedure analogous to that described in Example 4B, the product ofExample 15F (192 mg, 0.66 mmol) was coupled with (1S,2S,3R,5S)pinanediolleucine boronate trifluoroacetate salt (274 mg, 0.73 mmol) in thepresence of TBTU (277 mg, 0.73 mmol) to provide the title compound (286mg).

H. cis-3-Phenyl-D,L-proline-L-leucine boronic acid hydrochloride salt

The product of Example 15G (262 mg) was dissolved in CH₂Cl₂ (5 mL) andtreated at 0° C. with 4N HCl-dioxane (4 mL). After 2 h, the reactionmixture was concentrated to dryness, and the residue was treated withisobutylboronic acid (66 mg, 0.64 mmol) according to the proceduredescribed in Example 3B to provide the title compound (71 mg) as a whitesolid.

Example 17 trans-3-Phenyl-D,L-proline-L-leucine boronic acidhydrochloride salt [MG-363] A. N-Boc-trans-3-Phenyl-L-proline

By a procedure analogous to that described in Example 1A,N-acetyl-trans-3-phenyl-D,L-proline (prepared as described in Example15D; 1.5 g, 6.44 mmol) was coupled with (S)-a-methylbenzylamine (0.92mL, 7.08 mmol) in the presence of EDC (1.26 g, 7.08 mmol) and HOBT 9956mg, 7.08 mmol). The diastereomeric products were separated by flashchromatography (elution with 1.5–2.5% HOAc-EtOAc). Fractionscorresponding to the slower eluting band were concentrated to provide aclear, colorless oil (913 mg).

The oil (900 mg, 2.68 mmol) was dissolved in a mixture of HOAc (7 mL)and 8N HCl and the mixture was heated at reflux for 18 h. The mixturewas concentrated to dryness. The residue was dissolved in water (30 mL),washed with EtOAc, and again concentrated to dryness.

The residue was redissolved in 1:1 water-1,4-dioxane (15 mL) and treatedwith tert-butyl pyrocarbonate (1.13 g, 5.20 mmol) by a procedureanalogous to that described in Example 15F to provide the title compound(574 mg) as a white solid.

B. trans-3-Phenyl-L-proline-L-leucine boronic acid hydrochloride salt

By procedures analogous to those described in Examples 15G–H, theproduct of Example 16A (332 mg, 1.14 mmol) was coupled with(1S,2S,3R,5S)-pinanediol leucine boronate trifluoroacetate salt (452 mg,1.20 mmol) and deprotected to provide the title compound (101 mg) as awhite solid.

Example 18 Kinetic Experiments

Table II summarizes results from kinetic experiments that measured theinhibition of the 20S proteasome by compounds having the formula ofcompound (1) or (2). P, AA¹, AA², AA³, and Z¹ and Z² represent thestructures present on formula (1) or (2). The protocol for the kineticassay described in Tables II–V is as described in Rock et al., Cell78:761–771 (1994). In these tables, K_(i) values are reported, which aredissociation constants for the equilibrium that is established whenenzyme and inhibitor interact to form the enzyme:inhibitor complex. Thereactions were performed using SDS-activated 20S proteasome from rabbitmuscle. The substrate used was Suc-LLVY-AMC.

TABLE II Inhibition of the 20S Proteasome by Boronic Ester and AcidCompounds P-AA¹-AA²-AA³-B(Z¹)(Z²) 20S Compound P^(d) AA¹ AA^(2b) AA^(3c)Z¹, Z² K_(i) (nM) MG-261 Cbz L-Leu L-Leu L-Leu pinane diol 0.032 MG-262Cbz L-Leu L-Leu L-Leu (OH)₂ 0.035 MG-264 Cbz — L-Leu L-Leu pinane diol119.00 MG-267 Cbz — L-Nal L-Leu pinane diol 0.100 MG-268 Cbz(N-Me) —L-Leu L-Leu (OH)₂ 998.00 MG-270 Cbz — L-Nal L-Leu (OH)₂ 0.083 MG-272 Cbz— D-(2-Nal) L-Leu (OH)₂ 34.0 MG-273 Morph — L-Nal L-Leu (OH)₂ 0.18MG-274 Cbz — L-Leu L-Leu (OH)₂ 3.0 MG-278 Morph L-Leu L-Leu L-Leu (OH)₂0.14 MG-282 Cbz — L-His L-Leu (OH)₂ 25.0 MG-283 Ac L-Leu L-Leu L-Leu(OH)₂ 0.46 MG-284

— — L-Leu (OH)₂ 1,200 MG-285 Morph — L-Trp L-Leu (OH)₂ 3.0 MG-286 Morph— L-Nal L-Leu diethanol- 0.15 amine MG-287 Ac — L-Nal L-Leu (OH)₂ 0.13MG-288 Morph — L-Nal D-Leu (OH)₂ 72.5 MG-289 Ms — L-(3-Pal) L-Leu (OH)₂6.3 MG-290 Ac — L-(3-Pal) L-Leu (OH)₂ 5.4 MG-291 Ms — L-Nal L-Leudiethanol- 0.28 amine MG-292 Morph —

L-Leu (OH)₂ 6.0 MG-293 Morph — D-Nal D-Leu (OH)₂ 2,300 MG-294 H —L-(3-Pal) L-Leu (OH)₂ 152 MG-295 Ms — L-Trp L-Leu (OH)₂ 5.8 MG-296(8-Quin)-SO₂ — L-Nal L-Leu (OH)₂ 1.7 MG-297 Ts — L-Nal L-Leu (OH)₂ 0.17MG-298 (2-Quin)-C(O) — L-Nal L-Leu (OH)₂ 0.075 MG-299(2-quinoxalinyl)-C(O) — L-Nal L-Leu (OH)₂ 0.14 MG-300 Morph — L-(3-Pal)L-Leu (OH)₂ 1.3 MG-301 Ac — L-Trp L-Leu (OH)₂ 1.3 MG-302 H — L-Nal L-Leu(OH)₂ 7.5 MG-303 H.HCl — L-Nal L-Leu (OH)₂ 3.9 MG-304 Ac L-Leu L-NalL-Leu (OH)₂ 0.022 MG-305 Morph — D-Nal L-Leu (OH)₂ 189 MG-306 Morph —L-Tyr-(O-Benzyl) L-Leu (OH)₂ 0.23 MG-307 Morph — L-Tyr L-Leu (OH)₂ 0.51MG-308 Morph — L-(2-Nal) L-Leu (OH)₂ 0.72 MG-309 Morph — L-Phe L-Leu(OH)₂ 0.82 MG-310 Ac —

L-Leu (OH)₂ 90 MG-312 Morph — L-(2-Pal) L-Leu (OH)₂ 6.3 MG-313Phenethyl-C(O) — — L-Leu (OH)₂ 42 MG-314 (2-Quin)-C(O) — L-Phe L-Leu(OH)₂ 0.19 MG-315 Morph —

L-Leu (OH)₂ 2.2 MG-316 H.HCl —

L-Leu (OH)₂ 22 MG-317 Morph — L-Nal L-Leu (OH)(CH₃) 99 MG-318 Morph —L-Nal L-Leu (CH₃)₂ 640 MG-319 H.HCl — L-Pro L-Leu (OH)₂ 20 MG-321 Morph— L-Nal L-Phe (OH)₂ 0.32 MG-322 Morph — L-homoPhe L-Leu (OH)₂ 2.2 MG-323Ac — — L-Leu (OH)₂ 850 MG-324

— — L-Leu (OH)₂ 2.0 MG-325 (2-Quin)-C(O) — L-homoPhe L-Leu (OH)₂ 2.8MG-328 Bz — L-Nal L-Leu (OH)₂ 0.088 MG-329 Cyclohexyl-C(O) — L-Nal L-Leu(OH)₂ 0.03 MG-332 Cbz (N-Me) — L-Nal L-Leu (OH)₂ 0.95 MG-333 H.HCl(N-Me) — L-Nal L-Leu (OH)₂ 2.1 MG-334 H.HCl (N-Me) — L-Nal L-Leu (OH)₂1.1 MG-336 (3-Pyr)-C(O) — L-Phe L-Leu (OH)₂ 0.25 MG-337 H.HCl —

L-Leu (OH)₂ 230 MG-338 (2-Quin)-C(O) — L-(2-Pal) L-Leu (OH)₂ 1.4 MG-339H.HCl —

L-Leu (OH)₂ 1,600 MG-340 H —

L-Leu (OH)₂ 480 MG-341 (2-Pyz)-C(O) — L-Phe L-Leu (OH)₂ 0.6 MG-342 Bn —

— (OH)₂ 9,700 MG-343 (2-Pyr)-C(O) — L-Phe L-Leu (OH)₂ 0.42 MG-344 Ac —

L-Leu (OH)₂ 51 MG-345 Bz — L-(2-Pal) L-Leu (OH)₂ 0.76 MG-346Cyclohexyl-C(O) — L-(2-Pal) L-Leu (OH)₂ 1.1 MG-347 (8-Quin)-SO₂ —L-(2-Pal) L-Leu (OH)₂ 29 MG-348 H.HCl —

L-Leu (OH)₂ 21 MG-349 H.HCl —

L-Leu (OH)₂ 18 MG-350

— L-Phe L-Leu (OH)₂ 0.14 MG-351 H.HCl — L-(2-Pal) L-Leu (OH)₂ 32 MG-352Phenethyl-C(O) — L-Phe L-Leu (OH)₂ 0.15 MG-353 Bz — L-Phe L-Leu (OH)₂0.15 MG-354 (8-Quin)-SO₂ —

L-Leu (OH)₂ 28 MG-356 Cbz — L-Phe L-Leu (OH)₂ 0.13 MG-357 H.HCl —

L-Leu (OH)₂ 23 MG-358 (3-Furanyl)-C(O) — L-Phe L-Leu (OH)₂ 0.17 MG-359H.HCl —

L-Leu (OH)₂ 5.5 MG-361 (3-Pyrrolyl)-C(O) — L-Phe L-Leu (OH)₂ 0.14 MG-362

— — L-Leu (OH)₂ 6,400 MG-363 H.HCl —

L-Leu (OH)₂ 3.45 MG-364 Phenethyl-C(O) — — L-Leu (OH)₂ 1,500 MG-366H.HCl —

L-Leu (OH)₂ 45.2 MG-368 (2-Pyz)-C(O) — L-(2-Pal) L-Leu (OH)₂ 5.6 MG-369H.HCl —

L-Leu (OH)₂ 24.2 MG-380 (8-Quin)SO₂ — L-Phe L-Leu (OH)₂ 4.4 MG-382(2-Pyz)-C(O) — L-(4-F)-Phe L-Leu (OH)₂ 0.95 MG-383 (2-Pyr)-C(O) —L-(4-F)-Phe L-Leu (OH)₂ 0.84 MG-385 H.HCl —

L-Leu (OH)₂ 23 MG-386 H.HCl —

L-Leu (OH)₂ 92 MG-387 Morph —

L-Leu (OH)₂ 0.2 ^(a)Cbz = carbobenzyloxy; MS = methylsulfonyl; Morph =4-morpholinecarbonyl; (8-Quin)-SO₂ = 8-quinolinesulfonyl; (2-Quin)-C(O)= 2-quinolinecarbonyl; Bz = benzoyl; (2-Pyr)-C(O) = 2-pyridinecarbony;(3-Pyr)-C(O) = 3-pyridinecarbonyl; (2-Pyz)-C(O) = 2-pyrazinecarbonyl.^(b)Nal = β-(1-naphthyl)alanine; (2-Nal) = β-(2-naphthyl)alanine;(2-Pal) = β-(2-pyridyl)alanine; (3-Pal) = β-(3-pyridyl)alanine; homoPhe= homophenylalanine; (4-F)-Phe = (4-fluorophenyl)alanine. ^(c)B(Z¹)(Z²)takes the place of the carboxyl group AA³.

In Table III, P, AA¹, AA², AA³, and X are substituents of the generalformula: P-AA¹-AA²-AA³-X

Table III demonstrates that dipeptide boronic acids have lower K_(i)values than the corresponding dipeptide aldehydes.

TABLE III Comparison of Dipeptide Boronic Acids to Dipeptide AldehydesCpd. P AA¹ AA² AA³ X 20S K_(i) (nM) MG-105 Z — L-Leu L-Leu CHO 15,000MG-274 Z — L-Leu L-Leu B(OH)₂ 3.0

In Table IV, P, AA¹, AA², AA³, and X are substituents of the generalformula: P-AA¹-AA²-AA³-X.

Table IV demonstrates the markedly superior selectivity for the 20Sproteasome over other proteases, e.g. Cathepsin B, exhibited by theboronic esters/acids as compared to the peptide aldehydes.

TABLE IV Inhibition of the 20S Proteasome by Boronic Ester and AcidCompounds P-AA¹-AA²-AA³-X 20S Cathepsin B Compound P AA¹ AA² AA³ X K_(i)(nM) K_(i) (nM) MG-154 Ac L-Leu L-Leu L-Leu CHO 66.0 5.0 MG-191 CbzL-Trp L-Leu L-Leu CHO 0.38 0.54 MG-262 Cbz L-Leu L-Leu L-Leu B(OH)₂0.035 6,100. MG-273

— L-Nal L-Leu B(OH)₂ 0.18 200,000 MG-296

— L-Nal L-Leu B(OH)₂ 1.7 4,000 MG-309

— L-Phe L-Leu B(OH)₂ 0.82 132,000 M-341

— L-Phe L-Leu B(OH)₂ 0.6 160,000

The selectivity of boronic acid inhibitors of the proteasome is furtherdemonstrated in Table V.

TABLE V Selectivity of Boronic Ester and Acid Inhibitors of the 20SProteasome Human Human Leukocyte Pancreatic 20S Elastase Cathepsin GChymotrypsin Compound K_(i) (nM) K_(i) (nM) K_(i) (nM) K_(i) (nM) MG-2620.03 15 55 7 MG-267 0.1 150 33,000 2,300 MG-296 1.7 36 9,200 75 MG-3090.82 7,000 4,800 465 MG-341 0.6 2,300 628 322

Example 19 Inhibition of Protein Degradation in C2C12 Cells

C2C12 cells (a mouse myoblast line) were labelled for 48 hrs with³⁵S-methionine. The cells were then washed and preincubated for 2 hrs inthe same media supplemented with 2 mM unlabelled methionine. The mediawas removed and replaced with a fresh aliquot of the preincubation mediacontaining 50% serum, and a concentration of the compound to be tested.The media was then removed and made up to 10% TCA and centrifuged. TheTCA soluble radioactivity was counted. Inhibition of proteolysis wascalculated as the percent decrease in TCA soluble radioactivity. Fromthis data, an EC₅₀ for each compound was calculated.

Data for compounds of formula (1) or (2) are presented in Table VI.

TABLE VI Inhibition of Protein Degradation in C2Cl2 Cells by BoronicEster and Acid Compounds P-AA¹-AA²-AA³-B(Z¹)(Z²) Compound P^(a) AA¹AA^(2b) AA^(3e) Z¹, Z² IC₅₀ (nM) MG-262 Cbz L-Leu L-Leu L-Leu (OH)₂ 280MG-270 Cbz — L-Nal L-Leu (OH)₂ 730 MG-272 Cbz — D-(2-Nal) L-Leu (OH)₂6,000 MG-273 Morph — L-Nal L-Leu (OH)₂ 140 MG-274 Cbz — L-Leu L-Leu(OH)₂ 340 MG-278 Morph L-Leu L-Leu L-Leu (OH)₂ 7,500 MG-282 Cbz — L-HisL-Leu (OH)₂ 64,000 MG-283 Ac L-Leu L-Leu L-Leu (OH)₂ 3,000 MG-285 Morph— L-Trp L-Leu (OH)₂ 2,400 MG-286 Morph — L-Nal L-Leu diethanolamine 95MG-287 Ac — L-Nal L-Leu (OH)₂ 106 MG-289 Ms — L-(3-Pal) L-Leu (OH)₂10,830 MG-290 Ac — L-(3-Pal) L-Leu (OH)₂ 10,240 MG-292 Morph —

L-Leu (OH)₂ 11,320 MG-296 (8-Quin)-SO₂ — L-Nal L-Leu (OH)₂ 738 MG-298(2-Quin)-C(O) — L-Nal L-Leu (OH)₂ 230 MG-299 (2-Quinoxalinyl)-C(O) —L-Nal L-Leu (OH)₂ 280 MG-301 Ac — L-Trp L-Leu (OH)₂ 1,300 MG-302 H —L-Nal L-Leu (OH)₂ 270 MG-303 H.HCl — L-Nal L-Leu (OH)₂ 340 MG-304 AcL-Leu L-Nal L-Leu (OH)₂ 240 MG-306 Morph — L-Tyr-(O-Bn) L-Leu (OH)₂ 130MG-307 Morph — L-Tyr L-Leu (OH)₂ 4,800 MG-308 Morph — L-(2-Nal) L-Leu(OH)₂ 96 MG-309 Morph — L-Phe L-Leu (OH)₂ 210 MG-312 Morph — (L-(2-Pal)L-Leu (OH)₂ 1,100 MG-313 Phenethyl-C(O) — — L-Leu (OH)₂ 3,500 MG-314(2-Quin)-C(O) — L-Phe L-Leu (OH)₂ 130 MG-315 Morph —

L-Leu (OH)₂ 340 MG-316 H.HCl —

L-Leu (OH)₂ 21,000 MG-319 H.HCl — L-Pro L-Leu (OH)₂ 14,000 MG-321 Morph— L-Nal L-Phe (OH)₂ 2,400 MG-322 Morph — L-homoPhe L-Leu (OH)₂ 380MG-325 (2-Quin)-C(O) — L-homoPhe L-Leu (OH)₂ 1,100 MG-328 Bz — L-NalL-Leu (OH)₂ 69 MG-329 Cyclohexyl-C(O) — L-Nal L-Leu (OH)₂ 48 MG-322 Cbz(N-Me) — L-Nal L-Leu (OH)₂ 950 MG-333 H.HCl (N-Me) — L-Nal L-Leu (OH)₂220 MG-334 H.HCl (N-Me) — L-Nal L-Leu (OH)₂ 320 MH-336 (3-Pyr)-C(O) —L-Phe L-Leu (OH)₂ 100 MG-341 (2-Pyz)-C(O) — L-Phe L-Leu (OH)₂ 69 MG-343(2-Pyr)-C(O) — L-Phe L-Leu (OH)₂ 57 MG-345 Bz — L-(2-Pal) L-Leu (OH)₂120 MG-346 Cyclohexyl-C(O) — L-(2-Pal) L-Leu (OH)₂ 150 MG-347(8-Quin)-SO₂ — L-(2-Pal) L-Leu (OH)₂ 13,000 MG-350

— L-Phe L-Leu (OH)₂ 160 MG-351 H.HCl — L-(2-Pal) L-Leu (OH)₂ 8,200^(a)Cbz = carbobenyloxy; Morph = 4-morpholinecarbonyl; (8-Quin)SO₂ =8-quinolinesulfonyl; (2-Quin)C(O) = 2-quinolinecarbonyl; Bz = benzoyl;(2-Pyr)-C(O) = 2-pyridinecarbonyl; (3-Pyr)-C(O) = 3-pyridinecarbonyl;(2-Pyz)-C(O) = 2-pyrazinecarbonyl. ^(b)Nal = β-(1-naphthyl)alanine;(2-Nal) = β-(2-naphthyl)alanine; (2-Pal) = β-(2-pyridyl)alanine; (3-Pal)= b-(3-pyridyl)alanine; homoPhe = homophenylalanine. ^(c)B(Z¹)(Z²) takesthe place of the carboxyl group of AA³.

Example 20 MG-273 Inhibits Corticosterone-Induced Cachexia in Rats

Rats were stabilized on a diet free from 3-methylhistidine and thenplaced in metabolic cages for collection of 24-hour urine samples. Aftertwo days of urine collections to determine basal 3-methylhistidineoutput, the rats were treated with daily subcutaneous injections ofcorticosterone (100 mg/kg). Starting on the second day of corticosteronetreatment, some of the rats were also treated with MG-273, administeredvia a subcutaneous osmotic pump at a dose rate of approximately 120μg/kg body weight/day. Control rats received vehicle only (25% DMSO/75%PEG (200)), administered in a similar fashion. FIG. 1 shows thattreatment with MG-273 reduced the urinary output of 3-methylhistidine,which was induced in response to corticosterone treatment.

Example 21 MG-273 Inhibits the Activation of NF-κB

This assay was performed as previously described (Palombella, et al.Cell, 78:773–785 (1994)). MG63 osteosarcoma cells were stimulated bytreatment with TNF-α for the designated times. Whole cell extracts wereprepared and analyzed by electrophoretic mobility shift assay using thePRDII probe from the human IFN-β gene promoter. FIG. 2 shows that NF-κBbinding activity was inhibited by pretreatment for 1 hour with MG 273.An aldehyde inhibitor of the proteasome, MG-132(Cbz-L-Leu-L-Leu-L-Leu-H), also inhibited NF-κB binding activity,whereas MG-102 (Ac-L-Leu-L-Leu-L-Met-H), which is inactive against the20S proteasome, did not inhibit NF-κB binding activity.

Example 22 MG-273 Inhibits Expression of Cell Adhesion Molecules on HUVECells

HUVECs in microtiter plates were exposed to the indicated concentrationsof inhibitor for 1 hour, prior to the addition of 100 U/mL TNF-α. Cellsurface binding assays were performed at 4° C., using saturatingconcentrations of monoclonal antibodies specific for the cell adhesionmolecules (Becton Dickenson) and fluorescent-conjugated F(ab′)₂ goatanti-murine IgG (Caltag Labs, San Francisco, Calif.). Fluorescentimmunoassays for E-selectin and I-CAM were performed at 4 hours, thosefor V-CAM at 16 hours. FIG. 3 shows that cell-surface expression I-CAM,V-CAM, and E-selectin on TNF-α stimulated HUVECs is significantlyinhibited by MG-273 at concentrations of 0.5 μM or above.

Example 23 Boronic Acid Compounds Block the DTH Response in Mice

Naive mice were sensitized by the application of 20 μL of a 0.5% (v/v)solution of 2,4-dinitrofluorobenzene in 4:1 acetone/olive oil to both ofthe rear limb footpads. This procedure is performed on two consecutivedays, which are referred to as days 0 and 1.

The efferent phase of the contact sensitivity response was elicited onday 5 by the application of 10 μL of a 0.2% (v/v) solution of2,4-dinitrofluorobenzene in 4:1 acetone/olive oil to both sides of theleft ear. The contralateral control ear was treated on both sides with10 μL of vehicle only. The mice were lightly anaesthetized for thisprocedure by the intraperitoneal (i.p.) injection of a mixture ofketamine (80 mg/kg, Henry Schein) and xylazine (16 mg/kg, Henry Schein).

Test compounds were administered orally as a suspension in 0.5%methylcellulose (4000 centipoises Fisher Scientific) 30 minutes prior tothe application of the challenge dose of 2,4-dinitrofluorobenzene to theears. The dose was delivered in a final volume of 0.5 mL using a 24gauge 1 inch malleable feeding needle with a 1.25 mm ball tip (RobozSurgical).

Approximately 18 hours after the challenge, ear swelling was determinedby measuring both the control and the experimental ear using a MitutoyoDigital micrometer. The absolute difference in thickness of theexperimental (left) ears vs. the control (right) ears was determined foreach treatment group. Efficacy was determined by comparing thisdifference in thickness to the difference calculated for the vehiclecontrol group. Test results are provided in Table VII.

TABLE VII Inhibition of the DTH Response in Mice Compound Dose (mg/kg) %Inhibition MG-296 50 60 MG-309 3 40 MG-341 3 90

All publications and U.S. patent applications mentioned hereinabove arehereby incorporated in their entirety by reference.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be appreciated by oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention and appended claims.

1. A compound having the formula (1a):

or a pharmaceutically acceptable salt thereof; wherein P is hydrogen oran amino group protecting moiety; A is zero; X² is —C(O)—NH—; R ishydrogen or C₁₋₈ alkyl; R² is —CH₂—R⁵; R³ is C₄ alkyl; R⁵ is aryl orcycloalkyl, wherein R⁵ is optionally substituted by one or twosubstituents independently selected from the group consisting of C₁₋₆alkyl, C₃₋₈ cycloalkyl, C₁₋₆alkyl(C₃₋₈)cycloalkyl, C₂₋₈ alkenyl, C₂₋₈alkynyl, cyano, amino, C₁₋₆ alkylamino, di(C₁₋₆)alkylamino, benzylamino,dibenzylamino, nitro, carboxy, carbo(C₁₋₆)alkoxy, trifluoromethyl,halogen, C₁₋₆ alkoxy, C₆₋₁₀ aryl, C₆₋₁₀ aryl(C₁₋₆)alkyl, C₆₋₁₀aryl(C₁₋₆)alkoxy, hydroxy, C₁₋₆ alkylthio, C₁₋₆ alkylsulfinyl, C₁₋₆alkylsulfonyl, C₆₋₁₀ arylthio, C₆₋₁₀ arylsulfinyl, C₆₋₁₀ arylsulfonyl,C₁₋₆ alkyl(C₆₋₁₀)aryl, and halo(C₆₋₁₀)aryl; Z¹ and Z² are eachindependently one of alkyl, hydroxy, alkoxy, or aryloxy, or together Z¹and Z² form a moiety derived from a dihydroxy compound having at leasttwo hydroxy groups separated by at least two connecting atoms in a chainor ring, said chain or ring comprising carbon atoms and, optionally, aheteroatom or heteroatoms which can be N, S, or O.
 2. The compound ofclaim 1, wherein R is hydrogen.
 3. The compound of claim 1, wherein R³is isobutyl.
 4. The compound of claim 1, wherein P is R⁷—C(O)— orR⁷—SO₂—, where R⁷ is one of alkyl, cycloalkyl, aryl, aralkyl,heteroaryl, heteroarylalkyl, or a saturated or partially unsaturatedheterocycle, wherein the ring portion of R⁷ is optionally substituted.5. The compound of claim 1, wherein P is R⁷—NH—C(O)— or R⁷—O—C(O)—,where R⁷ is one of alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, orheteroarylalkyl, wherein the ring portion of R⁷ is optionallysubstituted.
 6. The compound of claim 4 or 5, wherein R⁷ is anoptionally substituted aryl or aralkyl.
 7. The compound of claim 4 or 5,wherein R⁷ is an optionally substituted heteroaryl or heteroaralkyl. 8.The compound of claim 1, wherein R⁵ is an optionally substituted C₆₋₁₀aryl.
 9. The compound of claim 1, wherein R⁵ is phenyl.
 10. The compoundof claim 9, wherein Z¹ and Z² are both hydroxy.
 11. The compound ofclaim 9, wherein Z¹ and Z² together form a moiety derived from adihydroxy compound having at least two hydroxy groups separated by atleast two connecting atoms in a chain or ring, said chain or ringcomprising carbon atoms and, optionally a heteroatom or heteroatomsindependently selected from the group consisting of N, S, and O.
 12. Acompound having the formula (1a):

or a pharmaceutically acceptable salt thereof; wherein P is R⁷—C(O)— orR⁷—SO₂—, and R⁷ is an optionally substituted aryl or aralkyl; A is zero;X² is —C(O)—NH—; R is hydrogen; R² is benzyl; R³ is C₄ alkyl; and Z¹ andZ² are independently one of hydroxy, alkoxy, or aryloxy, or together Z¹and Z² form a moiety derived from a dihydroxy compound having at leasttwo hydroxy groups separated by at least two connecting atoms in a chainor ring, said chain or ring comprising carbon atoms and, optionally, aheteroatom or heteroatoms which can be N, S, or O.
 13. The compound ofclaim 12, wherein R⁷ is phenyl.
 14. A composition, which uponcombination with a physiologically acceptable saline carrier forms asolution suitable for intravenous, intramuscular or subcutaneousadministration to a patient, said solution comprising a compound of theformula (1a):

or a pharmaceutically acceptable salt thereof; wherein P is hydrogen oran amino group protecting moiety; A is zero; X² is —C(O)—NH—; R ishydrogen or C₁₋₈ alkyl; R² is —CH₂—R⁵; R³ is C₄ alkyl; R⁵ is aryl orcycloalkyl, wherein R⁵ is optionally substituted by one or twosubstituents independently selected from the group consisting of C₁₋₆alkyl, C₃₋₈ cycloalkyl, C₁₋₆alkyl(C₃₋₈)cycloalkyl, C₂₋₈ alkenyl, C₂₋₈alkynyl, cyano, amino, C₁₋₆ alkylamino, di(C₁₋₆)alkylamino, benzylamino,dibenzylamino, nitro, carboxy, carbo(C₁₋₆)alkoxy, trifluoromethyl,halogen, C₁₋₆ alkoxy, C₆₋₁₀ aryl, C₆₋₁₀ aryl(C₁₋₆)alkyl, C₆₋₁₀aryl(C₁₋₆)alkoxy, hydroxy, C₁₋₆ alkylthio, C₁₋₆ alkylsulfinyl, C₁₋₆alkylsulfonyl, C₆₋₁₀ arylthio, C₆₋₁₀ arylsulfinyl, C₆₋₁₀ arylsulfonyl,C₁₋₆ alkyl(C₆₋₁₀)aryl, and halo(C₆₋₁₀)aryl; Z¹ and Z² are both hydroxy.15. The composition of claim 14, wherein R is hydrogen.
 16. Thecomposition of claim 14, wherein R³ is isobutyl.
 17. The composition ofclaim 14, wherein P is R⁷—C(O)— or R⁷—SO₂—, where R⁷ is one of alkyl,cycloalkyl, aryl, aralkyl, heteroaryl, heteroarylalkyl, or a saturatedor partially unsaturated heterocycle, wherein the ring portion of R⁷ isoptionally substituted.
 18. The composition of claim 14, wherein P isR⁷—NH—C(O)— or R⁷—O—C(O)—, where R⁷ is one of alkyl, cycloalkyl, aryl,aralkyl, heteroaryl, or heteroarylalkyl, wherein the ring portion of R⁷is optionally substituted.
 19. The composition of claim 17 or 18,wherein R⁷ is an optionally substituted aryl or aralkyl.
 20. Thecomposition of claim 17 or 18, wherein R⁷ is an optionally substitutedheteroaryl or heteroaralkyl.
 21. The composition of claim 14, wherein R⁵is an optionally substituted C₆₋₁₀ aryl.
 22. The composition of claim14, wherein R⁵ is phenyl.
 23. A composition, which upon combination witha physiologically acceptable saline carrier forms a solution suitablefor intravenous, intramuscular or subcutaneous administration to apatient, said solution comprising a compound of the formula (1a):

or a pharmaceutically acceptable salt thereof; wherein P is R⁷—C(O)— orR⁷—SO₂—, and R⁷ is an optionally substituted aryl or aralkyl; A is zero;X² is —C(O)—NH—; R is hydrogen; R² is benzyl; R³ is C₄ alkyl; and Z¹ andZ² are both hydroxy.
 24. The composition of claim 23, wherein R⁷ isphenyl.